Polymorphisms of the BLyS gene and use in diagnostic methods

ABSTRACT

The present invention provides an isolated polynucleotide comprising at least one polymorphic nucleotide sequence, for example, the major alleles of the SNPs described as rs12583006, rs1224141, and rs1248930 and diagnostic assays for detecting the presence of these polymorphism associated with a condition associated with BLyS activity, such as hematological malignancy including B cell malignancies. The diagnostic assays are useful in predicting an individual&#39;s likelihood of developing a condition associated with BLyS activity, such as hematological malignancies, and for methods for treating an individual clinically diagnosed with a condition associated with BLyS activity, such as prediction of a patient&#39;s likelihood to respond to a specific drug treatment. The invention also provides an array of nucleic acid molecules immobilized on a solid surface, where at least one of the nucleic acid molecules comprises a BLyS polymorphic nucleic acid molecule. The nucleic acid arrays of the invention allow rapid detection of hybridizing nucleic acid-molecules, in a nucleic acid sample from an individual, of a BLyS polymorphism associated with hematological malignancy.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 60/991,509, filed Nov. 30, 2007, which is herein incorporated byreference.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under CA092104 andCA097274 awarded by the National Cancer Institute. The government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

B lymphocyte stimulator (BLyS) is a tumor necrosis factor (TNF) familymember critical for maintenance of normal B cell development andhomeostasis. It has been described by multiple names including BLyS(Moore et al. Science 1999; 285:260-263); BAFF (Schneider et al. J ExpMed 1999; 189:1747-1756); THANK (Mukhopadhyay et al. J Biol Chem 1999;274:15978-15981); and TALL-1 (Shu et al. J Leukoc Biol 1999;65:680-683). This ligand binds to three receptors in the TNF family:transmembrane activator and CAML interactor (TACI), B cell maturationantigen (BCMA) and BAFF receptor (BAFF-R) (Gross et al. Nature 2000;404:995-999 and Thompson et al. Science 2001; 293:2012-2013). In normalanimals, BLyS is expressed by monocytes, macrophages, dendritic cells,neutrophils, and radiation-resistant (non-myeloid) cells hypothesized tobe stromal cells of lymphoid organs (reviewed in Schneider, Curr OpImmunol 2005; 17:282-289). BLyS production has been shown to beregulated by various cytokines including interferon gamma (IFN-γ),granulocyte and macrophage colony stimulating factor (GM-CSF) andinterleukin 10 (IL-10) (Scapini et al. J Exp Med 2003; 297-302 andNardelli et al. Blood 2001; 97:198-204).

Because overexpression of BLyS in transgenic animals resulted inautoimmune-like symptoms, reminiscent of systemic lupus erythematosus(SLE) and Sjörgens Syndrome (see Gross et al., supra.), investigationinto BLyS levels in the clinic initially focused on autoimmune diseases.Genetic analysis of patients with SLE and rheumatoid arthritis (RA)discovered certain polymorphisms in the gene structure, although none ofthe polymorphisms could be associated in a statistically significant waywith susceptibility to either disease (Kawasaki et al. Gen & Immun 2002;3:424-429). However, one mutation (C→T at position −871) was shown to beassociated with increased anti-Sm antibody and elevated monocyte BLySlevels.

Later work has focused on the role of BLyS in hematologicalmalignancies. BLyS has been shown to be expressed in malignant B cellssuch as B chronic lymphocytic leukemia (B-CLL) cells Novak et al. Blood2002; 100: 2973-2979), multiple myeloma (MM) (Novak et al. Blood 2004;103:689-694), B-cell lymphoma (He et al. J Immunol 2004; 172:3268-3279), and non-Hodgkin's lymphoma (HL) (Briones et al. Exp Hematol2002; 30:135-141). For NHL, levels of BLyS and its receptors has beencorrelated with disease activity and patient outcome (Novak et al. Blood2004; 104: 2247-2253). Overexpression of this ligand has also beendemonstrated in patients with Waldenstrom's Macroglobulinema (WM), afurther B cell malignancy (Elsawa et al., Blood 2006; 107:2882-2888).

An additional area where BLys levels have been measured is in T cells,particularly in association with T cell related diseases, including Tcell malignancies and autoimmune diseases with strong T cell associationsuch as multiple sclerosis (Krumbholz et al. 2005; J Exp Med 201:195)and rheumatoid arthritis (Matsumoto et al. Science 1999; 286:1732-1753). Several groups have examined the significant effect of BLySon T cell stimulation (Ng et al. J Immunol 2004; 173: 807 and Huard etal. Int Immunity 2004; 16: 467). This observation was broadened toinclude Th1 and Th2 responses and T cell mediated inflammatory reactions(Sutherland et al. J Immunol 2005; 174: 5537-5544). Thus, it appearsthat BLyS may play a general role in hematological cancers whetheroriginating from B cells or T cells.

There remains a need in the art for further identification of geneticpolymorphisms of the BLyS gene or the sequences that control itsexpression that are statistically associated with hematologicalmalignancies, such as B-cell malignancies. Such information is importantfor identifying individuals who have a propensity toward developing suchhematological malignancies and for identifying new therapeutic agentsfor the treatment of these diseases. The present invention addressesthis need by providing a polymorphism associated with both high BLySlevels and hematological cancers, and diagnostic tests determining thepresence of this polymorphism.

SUMMARY OF THE INVENTION

The present invention provides isolated polypeptides comprisingpolymorphic nucleotide sequences of the BLyS gene identified asrs12583006, rs1224141, and rs12428930 (rs283296). Each of these singlenucleotide polymorphisms (SNPs) occurs within intron sequences of theBLyS gene. The present invention provides a polypeptide comprising allthree of these SNP sequences, in particular the major allele of each ofthese sequences. The polymorphic polypeptides of the present inventionresult in higher levels of BLyS polypeptide being produced thusresulting in BLyS activity. Other polymorphisms of the present inventionmay be located in the coding or non-coding portion of the gene. In someembodiments, the polymorphisms are associated with hematologicalmalignancies. In some of these embodiments, the hematologicalmalignancies are B cell malignancies, such as B-cell non-Hodgkinlymphoma (NHL). Isolated polymorphisms comprising one or morepolymorphisms of the BLyS gene that are associated with hematologicalmalignancies, as well as other tests characteristic of hematologicalmalignancies as described fully below, are useful in diagnostic assays.

The present invention provides an isolated polynucleotide comprising apolymorphic nucleotide sequence from a B-lymphocyte stimulator (BLyS)gene. The polymorphism may be in a coding or non-coding portion of thegene. In some embodiments, polymorphisms are associated with a conditionassociated with BLyS activity. In particular, the SNPs identified asrs12583006, rs1224141, and rs12428930 (rs283296) are reported herein asstatistically identified with conditions associated with BLyS activity,such as NHL. Even more particularly, the major allele of each of theseSNPs are associated with an increased chance of B cell malignancies suchas NHL, i.e., rs12583006 as a thymidine (T), rs1224141 as a thymidine(T), and rs12428930 (rs283296) as an adenine (A). Isolatedpolynucleotides comprising one or more polymorphisms in a BLyS gene suchas these that are associated with a condition associated with BLySactivity are useful in diagnostic assays. Isolated polynucleotidescomprising all three of these polymorphisms are particularly associatedwith an increased risk of B cell malignancies.

Accordingly, the invention further provides diagnostic assays fordetecting the presence in a nucleic acid sample of a polymorphism in aBLyS gene that is associated with a condition associated with BLySactivity, such as hematological malignancy including B cellmalignancies. Diagnostic assays are useful in predicting an individual'slikelihood of developing a condition associated with BLyS activity, suchas hematological malignancies. Thus, the invention further providesmethods of detecting a propensity in an individual of developing acondition associated with BLyS activity, such as hematologicalmalignancies. The invention further provides methods for geneticallydiagnosing in an individual a condition associated with BLyS activity,such as hematological malignancy. These methods generally involvedetecting in a nucleic acid sample derived from an individual a BLySpolymorphism associated with a condition associated with BLyS activity.In some embodiments, diagnostic assays are conducted using a microarraycomprising a BLyS polymorphic nucleic acid molecule.

Detection of BLyS polymorphisms associated with a condition associatedwith BLyS activity, such as hematological malignancy, allows selectionof a treatment plan that is most likely to be effective in treating thecondition. Thus, the invention further provides methods for treating anindividual clinically diagnosed with a condition associated with BLySactivity, generally comprising detecting a BLyS polymorphism associatedwith a condition associated with BLyS activity, and selecting atreatment plan that is most effective for individuals clinicallydiagnosed as having a condition associated with BLyS activity. Detectionof BLyS polymorphisms associated with a condition associated with BLySactivity also allows one to predict a patient's likelihood to respond toa specific drug treatment. Thus, the invention further provides methodsof predicting a patient's likelihood to respond to a specific drugtreatment for a condition associated with BLyS activity, such ashematological malignancy.

The invention further provides an array of nucleic acid moleculesimmobilized on a solid surface, where at least one of the nucleic acidmolecules comprises a BLyS polymorphic nucleic acid molecule. Thenucleic acid arrays of the invention allow rapid detection ofhybridizing nucleic acid-molecules, in a nucleic acid sample from anindividual, of a BLyS polymorphism associated with hematologialmalignancy.

These and other aspects of the invention will become apparent to thosepersons skilled the art upon reading the details of the invention asmore fully described below.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides polymorphisms in the human BLyS gene thatare associated with hematological malignancies, in particular B-cellmalignancies, such as NHL, and methods of using nucleic acid moleculescomprising the polymorphisms. The invention is based on the finding thatpolymorphisms in the intron region of the BLyS gene appear associatedwith the occurrence of NHL. Specifically, having a high risk allele atthese three polymorphisms particularly increases the risk for diffuselarge B-cell lymphoma and follicular lymphoma (grades I, II, or III)types of NHL. In each case, the high risk variant for the threeidentified SNPs was the major allele: for rs12583006 (T); rs1224141 (T);and re12428930 (A). This observation allows development of diagnosticassays to detect the presence of polymorphisms in an individual, whichpolymorphisms are associated with hematological malignancies, includingB-cell malignancies, such as NHL, and therefore may predict thelikelihood that an individual will develop a condition such ashematological malignancies, in particular, B-cell malignancies such asNHL, and provide insight as to the likely course of the disease upon itsdevelopment. Although these polymorphisms are found within introns ofthe BLyS gene, it is known in the art that such sequences can alter theexpression level of genes, see, for example, the increased expressiondriven by the CMV promoter when the sequences of intron A are present ina heterologous gene expression system (as described in thespecifications of U.S. Pat. Nos. 5,591,639 and 5,658,759). Without beingbound by theory, it is possible that the SNPs described herein aresimilarly associated with altered levels of transcription and/ortranslation of the BLyS protein as significantly higher levels of BLySwere measured in patients carrying such polymorphisms in their genomeand thus these polymorphisms result in an increased risk in thedevelopment of B cell malignancies. This observation leads to thepolynucleotides, polypeptides, and diagnostic tests of the presentinvention.

Before the present invention is described, it is to be understood thatthis invention is not limited to particular embodiments described, assuch may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “apolymorphism includes a plurality of such polymorphisms, reference to “anucleic acid molecule” includes a plurality of such nucleic acidmolecules, and reference to “the method” includes reference to one ormore methods, method steps, and equivalents thereof known to thoseskilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Definitions

As used herein, the term “BLyS gene” is intended to generically refer toboth the wild-type and variant forms of the sequence, unlessspecifically denoted otherwise. As it is commonly used in the art, theterm “gene” is intended to refer to the genomic region encompassing 5′untranslated region(s) (UTR), exons, introns, and 3′ UTR. Individualsegments may be specifically referred to, e.g. promoter, coding region,etc. Combinations of such segments that provide for a complete BLySprotein may be referred to generically as a protein coding sequence. Thenucleotide sequences of BLyS mRNA are publicly available throughGenBank: Accession No. NM 052945 (human BLyS mRNA). Provided is thehuman cDNA sequence as SEQ ID NO: 1, the human protein sequence as SEQID NO:2. Also encompassed are its human variants disclosed as AccessionNos. 003808, 172087, 172088. Further, Accession No. 033622 (mouse BLySmRNA) is contemplated. Provided is the mouse cDNA as SEQ ID NO:3,protein sequence as SEQ ID NO:4.

The term “polymorphism”, as used herein, refers to a difference in thenucleotide or amino acid sequence of a given region as compared to anucleotide or amino acid sequence in a homologous-region of anotherindividual, in particular, a difference in the nucleotide of amino acidsequence of a given region which differs between individuals of the samespecies. A polymorphism is generally defined in relation to a referencesequence. Polymorphisms include single nucleotide differences,differences in sequence of more than one nucleotide, and single ormultiple nucleotide insertions, inversions and deletions; as well assingle amino acid differences, differences in sequence of more than oneamino acid, and single or multiple amino acid insertions, inversions,and deletions. In particular, three polymorphisms of interest have beenidentified as rs12583006, rs1224141, and rs12428930 (rs283296) and canbe viewed in the single nucleotide polymorphism section of the NCBIwebsite and the BLyS sequences carrying the major alleles are disclosedas SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively in thepresent specification. It is noted that the major alleles of all threeof these SNPs are those associated with higher risk of developing B cellmalignancies, whereas the minor alleles are associated with lower riskof developing B cell malignancies.

As used herein, the term “polymorphic BLyS nucleic acid molecule” refersto a polynucleotide derived from a BLyS gene, which polynucleotidecomprises one or more polymorphisms when compared to a reference BLySpolynucleotide sequence. A polymorphism present in a polymorphic BLySnucleic acid molecule may be one that is associated with B-cellmalignancies, such as NHL.

An “allele” in relation to single nucleotide polymorphisms is thepresence of a particular nucleotide at a particular genomic location.Alleles can be classified as “high risk alleles” if the presence of aparticular nucleotide at a specific location is associated with anincreased risk of developing a particular disease. Alleles can also beclassified as “low risk alleles” if the presence of a particularnucleotide at a specific location does not appear to be associated withan increased risk of developing a particular disease.

The terms “polynucleotide” and “nucleic acid molecule” are usedinterchangeably herein to refer to polymeric forms of nucleotides of anylength. The polynucleotides may contain deoxyribonucleotides,ribonucleotides, and/or their analogs. Nucleotides may have anythree-dimensional structure, and may perform any function, known orunknown. The term “polynucleotide” includes single-, double-stranded andtriple helical molecules. “Oligonucleotide” generally refers topolynucleotides of between about 5 and about 100 nucleotides of single-or double-stranded DNA. However, for the purposes of this disclosure,there is no upper limit to the length of an oligonucleotide.Oligonucleotides are also known as oligomers or oligos and may beisolated from genes, or chemically synthesized by methods known in theart.

The following are non-limiting embodiments of polynucleotides: a gene orgene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. A nucleic acid molecule may alsocomprise modified nucleic acid molecules, such as methylated nucleicacid molecules and nucleic acid molecule analogs. Analogs of purines andpyrimidines are known in the art. Nucleic acids may be naturallyoccurring, e.g. DNA or RNA, or may be synthetic analogs, as known in theart. Such analogs may be preferred for use as probes because of superiorstability under assay conditions. Modifications in the native structure,including alterations in the backbone, sugars or heterocyclic bases,have been shown to increase intracellular stability and bindingaffinity. Among useful changes in the backbone chemistry arephosphorothioates; phosphorodithioates, where both of the non-bridgingoxygens are substituted with sulfur; phosphoroamidites; alkylphosphotriesters and boranophosphates. Achiral phosphate derivativesinclude 3′-O′-5′-S-phosphorothioate, 3′-S-5′-O-phosphorothioate,3′-CH2-5′-O-phosphonate and 3′-NH-5′-O-phosphoroamidate. Peptide nucleicacids replace the entire ribose phosphodiester backbone with a peptidelinkage.

Sugar modifications are also used to enhance stability and affinity. Theα-anomer of deoxyribose may be used, where the base is inverted withrespect to the natural β-anomer. The 2′-OH of the ribose sugar may bealtered to form 2′-O-methyl or 2′-O-allyl sugars, which providesresistance to degradation without comprising affinity.

Modification of the heterocyclic bases must maintain proper basepairing. Some useful substitutions include deoxyuridine fordeoxythymidine; 5-methyl-2′-deoxycytidine and 5-bromo-2′-deoxycytidinefor deoxycytidine. 5-propynyl-2′-deoxyuridine and5-propynyl-2′-deoxycytidine have been shown to increase affinity andbiological activity when substituted for deoxythymidine anddeoxycytidine, respectively.

The terms “polypeptide” and “protein”, used interchangebly herein, referto a polymeric form of amino acids of any length, which can includecoded and non-coded amino acids, chemically or biochemically modified orderivatized amino acids, and polypeptides having modified peptidebackbones. The term includes fusion proteins, including, but not limitedto, fusion proteins with a heterologous amino acid sequence, fusionswith heterologous and homologous leader sequences, with or withoutN-terminal methionine residues; immunologically tagged proteins; and thelike.

In the broadest sense, as used herein, the terms “a condition associatedwith BLyS activity,” and “a disease condition associated with BLySactivity,” refer to a condition or disease which results, directly orindirectly, from altered BLyS activity. “Altered BLyS activity,” as usedherein, includes one or more of the following: (1) BLyS biologicalactivity that is higher or lower than normal BLyS biological activity;(2) a level of BLyS mRNA in a cell that is higher or lower than thenormal level of BLyS mRNA for that cell type; and (3) a level of BLySpolypeptide that is higher or lower than the normal level of BLySpolypeptide. A condition associated with BLyS activity is also acondition or disease which is symptomatic of altered BLyS activity.“Normal BLyS biological activity,” “normal BLyS mRNA levels,” and“normal BLyS polypeptide levels” refer to BLyS activity that is in thenormal range for an individual of a given species, and which is notassociated with, or give rise to, a disease condition. A conditionassociated with BLyS activity includes, but is not limited to,hematological malignancies. “Hematological malignancies” are thosediseases generally having the properties of anaplasia, invasiveness, andmetastasis where the invasive cell is derived from the blood or bloodforming tissues, such as those classified in the World HealthOrganization Classification of Neoplastic Diseases of the Hematopoieticand Lymphoid Tissue (Jaffe et al. (Eds.) (2001). These diseases include,but are not limited to B-cell malignancies, T-cell malignancies, andnatural killer (K) cell malignancies. Among the B-cell malignanciesassociated with increased BLyS activities are B-CLL (includingfamilial), MM, B-cell lymphoma, and NHL. A representative type of acondition associated with increased BLyS activity is NHL.

Other B cell malignancies contemplated for the present invention are allvariants of the following hematological malignancies: precursor B-celllymphoblastic leukemia, B-cell prolymphocytic leukemia, immunocytoma,mantle cell lymphoma, follicular lymphoma, cutaneous follicularlymphoma, marginal zone B-cell lymphoma, hairy cell leukemia, diffuselarge B-cell lymphoma, mediastinal large B-cell lymphoma, intravascularlarge B-cell lymphoma, Burkitt lymphoma, monoclonal gammopathy ofundetermined significance, plasma cell myeloma variants beyond multiplemyeloma such as Indolent myeloma and smoldering myeloma, plasmacytomas,heavy chain disease, immunoglobulin deposition diseases such as systemiclight chain disease and primary amyloidosis. Among the T-cell and NKcell malignancies contemplated for the present invention are allvariants of the following malignancies: precursor T-cell lymphoblasticleukemia, T-cell prolymphocytic leukemia, T-cell granular lymphocyticleukemia, aggressive NK cell leukemia, nasal and nasal-type NK/T celllymphoma, mycosis fungoides and Sezary syndrome, angioimmunoblasticT-cell lymphoma, peripheral T-cell lymphoma unspecified, such asLennert's, anaplastic large cell lymphoma, primary cutaneous CD-30positive T-cell lymphoproliferative disorders, subcutaneouspanniculitis-like T-cell lymphoma, intestinal T-cell lymphoma, andhepatosplenic gamma/delta T-cell lymphoma. Other hematologicalmalignancies contemplated within the scope of the present inventioninclude all variants of, but are not limited to, Hodgkin Lymphoma,immunodeficiency related-lymphoproliferative disorders,histiocytic/dendritic cell neoplasms and related disorders, acuteleukemias and myelodysplastic syndromes, and chronic myeloproliferativedisorders.

The terms “a propensity to develop a condition associated with BLySactivity,” as used herein, refers to a statistically significantincrease in the probability of developing measurable characteristics ofa condition associated with BLyS activity in an individual having aparticular genetic lesion(s) or polymorphism(s) compared with theprobability in an individual lacking the genetic lesion or polymorphism.

A “substantially isolated” or “isolated” polynucleotide is one that issubstantially free of the sequences with which it is associated innature. By substantially free is meant at least 50%, preferably at least70%, more preferably at least 80%, and even more preferably at least 90%free of the materials with which it is associated in nature. As usedherein, an “isolated” polynucleotide also refers to recombinantpolynucleotides, which, by virtue of origin or manipulation: (1) are notassociated with all or a portion of a polynucleotide with which it isassociated in nature, (2) are linked to a polynucleotide other than thatto which it is linked in nature, or (3) does not occur in nature.

Hybridization reactions can be performed under conditions of different“stringency”. Conditions that increase stringency of a hybridizationreaction of widely known and published in the art. See, for example,Sambrook et al. (1989). Examples of relevant conditions include (inorder of increasing stringency): incubation temperatures of 25° C., 37°C., 50° C. and 68° C.; buffer concentrations of 10×SSC, 6×SSC, 1×SSC,0.1×SSC (where SSC is 0.15 M NaCl and 15 mM citrate buffer) and theirequivalents using other buffer systems; formamide concentrations of 0%,25%, 50%, and 75%; incubation times from 5 minutes to 24 hours; 1, 2, ormore washing steps; wash incubation times of 1, 2, or 15 minutes; andwash solutions of 6×SSC, 1×SSC, 0.1×SSC, or deionized water. Examples ofstringent conditions are hybridization and washing at 50° C. or higherand in 0.1×SSC (9 mM NaCl/0.9 mM sodium citrate).

“T_(m)” is the temperature in degrees Celsius at which 50% of apolynucleotide duplex made of complementary strands hydrogen bonded inanti-parallel direction by Watson-Crick base pairing dissociates intosingle strands under conditions of the experiment. T_(m) may bepredicted according to a standard formula, such as:

where [X⁺] is the cation concentration (usually sodium ion, Na⁺) inmol/L; (% G/C) is the number of G and C residues as a percentage oftotal residues in the duplex; (% F) is the percent formamide in solution(wt/vol); and L is the number of nucleotides in each strand of theduplex.

Stringent conditions for both DNA/DNA and DNA/RNA hybridization are asdescribed by Sambrook et al. Molecular Cloning, A Laboratory Manual, 2ndEd., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989, herein incorporated by reference. For example, see page 7.52 ofSambrook et al.

The term “host cell” includes an individual cell or cell culture whichcan be or has been a recipient of any recombinant vector(s) or isolatedpolynucleotide of the invention. Host cells include progeny of a singlehost cell, and the progeny may not necessarily be completely identical(in morphology or in total DNA complement) to the original parent celldue to natural, accidental, or deliberate mutation and/or change. A hostcell includes cells transfected or infected in vivo or in vitro with arecombinant vector or a polynucleotide of the invention. A host cellwhich comprises a recombinant vector of the invention is a “recombinanthost cell”.

The term “binds specifically,” in the context of antibody binding,refers to high avidity and/or high affinity binding of an antibody to aspecific polypeptide i.e., epitope of a polymorphic BLyS polypeptide.Antibody binding to an epitope on a specific polymorphic BLySpolypeptide (also referred to herein as “a polymorphic BLyS epitope”) ispreferably stronger than binding of the same antibody to any otherepitope, particularly those which may be present in molecules inassociation with, or in the same sample, as the specific polypeptide ofinterest, e.g., binds more strongly to a specific BLyS polymorphicepitope than to a different BLyS epitope so that by adjusting bindingconditions the antibody binds almost exclusively to the specific BLySpolymorphic epitope and not to any other BLyS epitope, and not to anyother BLyS polypeptide which does not comprise the polymorphic epitope.Antibodies which bind specifically to a polypeptide of interest may becapable of binding other polypeptides at a weak, yet detectable, level(e.g., 10% or less of the binding shown to the polypeptide of interest).Such weak binding, or background binding, is readily discernible fromthe specific antibody binding to the compound or polypeptide ofinterest, e.g. by use of appropriate controls. In general, antibodies ofthe invention which bind to a specific polymorphic BLyS polypeptide witha binding affinity of 10⁷ mole/l or more, preferably 10⁸ mole/liters ormore are said to bind specifically to the specific BLyS polymorphicpolypeptide. In general, an antibody with a binding affinity of 10⁶mole/liters or less is not useful in that it will not bind an antigen ata detectable level using conventional methodology currently used.

The terms “detectably labeled antibody,” “detectably labeledanti-polymorphic BLyS polypeptide,” “detectably labeled anti-BLySpolymorphic epitope,” or “detectably labeled anti-BLyS polymorphicpolypeptide fragment” refer to an antibody (or antibody fragment whichretains binding specificity for a polymorphic BLyS polypeptide orepitope), having an attached detectable label. The detectable label isnormally attached by-chemical conjugation, but where the label is apolypeptide, it could alternatively be attached by genetic engineeringtechniques. Methods for production of detectably labeled proteins arewell known in the art. Detectable labels may be selected from a varietyof such labels known in the art, including, but not limited to,radioisotopes, fluorophores, paramagnetic labels, enzymes (e.g.,horseradish peroxidase), or other moieties or compounds which eitheremit a detectable signal (e.g., radioactivity, fluorescence, color) oremit a detectable signal after exposure of the label to its substrate.Various detectable label/substrate pairs (e.g., horseradishperoxidase/diaminobenzidine, avidin/streptavidin,luciferase/luciferin)), methods for labeling antibodies, and methods forusing labeled antibodies are well known in the art (see, for example,Harlow and Lane, eds. (Antibodies: A Laboratory Manual (1988) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.)).

A “biological sample” encompasses a variety of sample types obtainedfrom an individual and can be used in a diagnostic or monitoring assay.The definition encompasses blood and other liquid samples of biologicalorigin, solid tissue samples such as a biopsy specimen or tissuecultures or cells derived therefrom and the progeny thereof. Thedefinition also includes samples that have been manipulated in any wayafter their procurement, such as by treatment with reagents,solubilization, or enrichment for certain components, such aspolynucleotides. The term “biological sample” encompasses a clinicalsample, and also includes cells in culture, cell supernatants, celllysates, serum, plasma, biological fluid, and tissue samples.

As used herein, the terms “treatment”, “treating”, and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse affectattributable to the disease. “Treatment”, as used herein, covers anytreatment of a disease in a mammal, particularly in a human, andincludes: (a) preventing the disease from occurring in a subject whichmay be predisposed to the disease but has not yet been diagnosed ashaving it; (b) inhibiting the disease, i.e., arresting its development;and (c) relieving the disease, i.e., causing regression of the disease.

The terms “individual,” “subject,” and “patient,” used interchangeablyherein, refer to a mammal, including, but not limited to, murines,simians, humans, mammalian farm animals, mammalian sport animals, andmammalian pets.

Isolated Polymorphic BLyS Nucleic Acid Molecules

The present invention provides isolated polynucleotides comprising oneor more BLys polymorphic nucleic acid molecules. In some embodiments,the polymorphism is one associated with conditions associated with BLySactivity, such as hematological malignancies. The isolatedpolynucleotides are useful in a variety of diagnostic methods. Isolatedpolymorphic BLyS nucleic acid molecules of the invention can be used inone or more of the following methods: a) screening assays; b) predictivemedicine (e.g., diagnostic assays, prognostics assays, monitoringclinical trials, and pharmacogenetics); and c) methods of treatment(e.g., therapeutic and prophylactic).

BLyS genes have been disclosed (see Moore et al.; Schneider et al.;Mukhopadhyay et al. and Shu et al. supra). The source of BLyS gene foruse in the present invention can be any mammalian BLyS gene. In general,for diagnostic assays, the animal source of the BLyS gene will be thesame species as the animal whose nucleic acid is being tested.

An isolated polymorphic BLyS nucleic acid molecule comprises one or moreBLyS polymorphisms. Specifically, the following three polymorphisms:rs12583006, rs1224141, and rs12428930 (rs283296) have been found asassociated with an increased risk of developing NHL. The sequences ofthese SNPs are reported within the BLyS genomic sequences labeled as SEQID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively, in the presentspecification. It should be noted that the high risk allele for theseSNPs in each case was the major allele. Specifically rs1258006 is T,rs1224141 is T, and rs12428930 (rs283296) is A. Highest risk for B cellmalignancy is found for individuals which carry the major allele in allthree of these polymorphisms, whereas the lowest risk is found for thoseindividuals who have the less common allele at all three SNP sites.

In some embodiments, the polymorphic BLyS sequence can comprises one ormore additional polymorphisms: (1) a G→A variation at or aboutnucleotide −1283 relative to the BLyS gene transcription start site; (2)a C→T variation at or about nucleotide −871 relative to the BLyS genetranscription start site; (3) a T→C variation at or about nucleotide−514 relative to the BLyS gene transcription start site; (4) a G→Cvariation at or about nucleotide −353 relative to the BLyS genetranscription start site; (5) a C→G variation at or about nucleotide 45(within intron 1 of the BLyS gene) and (6) a G→A variation at or aboutnucleotide 313 (causing a Ala105Thr change in the protein sequence). Theexact position of the aforementioned variants may vary from individualto individual or from species to species, e.g., by from 1 to about 10base pairs. Further polymorphisms include those described by Jiang etal. Immunogen 2001; 53: 810-814.

For some uses, e.g., in screening assays, BLyS polymorphic nucleic acidmolecules will be of at least about 15 nucleotides (nt), at least about18 nt, at least about 20 nt, or at least about 25 nt in length, andoften at least about 50 nt. Such small DNA fragments are useful asprimers for polymerase chain reaction (PCR), hybridization screening,etc. Larger polynucleotide fragments, e.g., at least about 50 nt, atleast about 100 nt, at least about 200 nt, at least about 300 nt, atleast about 500 nt, at least about 1000 nt, at least about 1500 nt, upto the entire coding region, or up to the entire coding region plus upto about 1000 nt 5′ and/or up to about 1000 nt 3′ flanking sequencesfrom a BLyS gene, are useful for production of the encoded polypeptide,promoter motifs, etc. For use in amplification reactions, such as PCR, apair of primers will be used. The exact composition of primer sequencesis not critical to the invention, but for most applications the primerswill hybridize to the subject sequence under stringent conditions, asknown in the art.

When used as a probe, an isolated polymorphic BLyS nucleic acid moleculemay comprise non-BLyS nucleotide sequences, as long as the additionalnon-BLyS nucleotide sequences do not interfere with the detection assay.A probe may comprise an isolated polymorphic BLyS sequence, and anynumber of non-BLyS nucleotide sequences, e.g., from about 1 bp to about1 kb or more.

For screening purposes, hybridization probes of the polymorphicsequences may be used where both forms are present, either in separatereactions, spatially separated on a solid phase matrix, or labeled suchthat they can be distinguished from each other. Assays (described below)may utilize nucleic acids that hybridize to one or more of the describedpolymorphisms.

Isolated polymorphic BLyS nucleic acid molecules of the invention may becoupled (e.g., chemically conjugated), directly or indirectly (e.g.,through a linker molecule) to a solid substrate. Solid substrates may beany known in the art including, but not limited to, beads, e.g.,polystyrene beads; chips, e.g., glass, SiO₂, and the like; plasticsurfaces, e.g., polystyrene, polycarbonate plastic multi-well plates;and the like.

Isolated polymorphic BLyS nucleic acid molecules can be obtained bychemical or biochemical synthesis, by recombinant DNA techniques, or byisolating the nucleic acids from a biological source, or a combinationof any of the foregoing. For example, the nucleic acid may besynthesized using solid phase synthesis techniques, as are known in theart. Oligonucleotide synthesis is also described in Edge et al. (1981)Nature 292:756; Duckworth et al. (1981) Nucleic Acids Res. 9:1691 andBeaucage and Caruthers (1981) Tet. Letters 22:1859. Followingpreparation of the nucleic acid, the nucleic acid is then ligated toother members of the expression system to produce an expression cassetteor system comprising a nucleic acid encoding the subject product inoperational combination with transcriptional initiation and terminationregions, which provide for expression of the nucleic acid into thesubject polypeptide products under suitable conditions.

Additional BLyS gene polymorphisms may be identified using any of avariety of methods known in the art, including, but not limited to SSCP,denaturing HPLC, and sequencing. Example 3 provides a description of howBLyS polymorphisms were identified using single strand conformationpolymorphism (SSCP) analysis and denaturing HPLC analysis. SSCP may beused to identify additional BLyS gene polymorphisms. In general, PCRprimers and restriction enzymes are chosen so as to generate products ina size range of from about 25 bp to about 500 bp, or from about 100 bpto about 250 bp, or any intermediate or overlapping range therein.

Polymorphic BLyS Polypeptides

The present invention provides isolated polymorphic BLyS polypeptides.Isolated polymorphic BLyS polypeptides are useful in assays to screenfor agents that modify a biological activity of a BLyS polypeptide.

The term “polymorphic BLyS polypeptide” encompasses an amino acidsequence encoded by an open reading frame (ORF) of a known BLySpolynucleotide, including the full-length native polypeptide andfragments thereof, particularly biologically active fragments and/orfragments corresponding to functional domains, e.g. a region or domainhaving biological activity, etc.; antigenic fragments thereof, andincluding fusions of the subject polypeptides to other proteins or partsthereof. The amino acid sequences of BLyS polypeptides have beendisclosed. See e.g. Moore et al., supra. A polymorphism in a BLySpolypeptide is generally defined relative to a reference sequence.

As used herein, “polymorphic BLyS polypeptide” refers to an amino acidsequence of a recombinant or non-recombinant polypeptide having an aminoacid sequence of i) a native polymorphic BLyS polypeptide, ii) afragment of a polymorphic BLyS polypeptide, iii) polypeptide analogs ofa polymorphic BLyS polypeptide, iv) variants of a polymorphic BLySpolypeptide; v) an immunologically active fragment of a polymorphic BLySpolypeptide; and vi) fusion proteins comprising a polymorphic BLySpolypeptide. Polymorphic BLyS polypeptides of the invention can beobtained from a biological sample, or from any source whether natural,synthetic, semi-synthetic or recombinant.

The term “polymorphic BLyS polypeptide” encompasses a polypeptidecomprising from at least about 5 amino acids, at least about 10 aminoacids, at least about 15 amino acids, at least about 25 amino acids, atleast about 50 amino acids, at least about 75 amino acids, at leastabout 100 amino acids, at least about 200 amino acids, at least about300 amino acids, at least about 400 amino acids, or up to the entirepolypeptide of a polymorphic BLyS polypeptide. In some embodiments, apolymorphic BLyS polypeptide exhibits biological activity, e.g., thepolypeptide causes proliferation of B-cells and production ofimmunoglobulin in an in vitro assay. Other assays for BLyS biologicalactivity are known in the art and can be used to determine whether apolymorphic BLyS polypeptide exhibits biological activity and, ifdesired, to quantitate BLyS biological activity. BLyS biological assaysare described in various publications, e.g., Moore et al., supra.

Polymorphic BLyS polypeptides of the invention may be part of a fusionprotein. Suitable fusion partners (e.g., a non-BLyS polypeptide, or“heterologous polypeptide”) include, but are not limited to, aheterologous polypeptide that provides for immunological recognition,e.g., an epitope tag; a heterologous polypeptide that provides for adetectable signal, e.g., a green fluorescent protein (GFP),β-galactosidase, and the like; a heterologous polypeptide that providesfor a catalytic function; and a heterologous polypeptide thatfacilitates entry into a cell. The fusion partner can be coupledin-frame to the N-terminus, the C-terminus, or both of the polymorphicBLyS polypeptide, using standard methods for synthesis of polypeptides,or using recombinant methods.

Polymorphic BLyS polypeptides of the invention can be obtained by anyknown method, or a combination of such methods, including isolation fromnatural sources; production by chemical synthesis; and production bystandard recombinant techniques.

Polymorphic BLyS polypeptides can be isolated from a biological source,using affinity chromatography, e.g., using antibodies specific for aBLyS polypeptide are immobilized on a solid support. The polypeptidesmay be expressed in prokaryotes or eukaryotes in accordance withconventional ways, depending upon the purpose for expression. For largescale production of the protein, a unicellular organism, such as E.coli, B. subtilis, S. cerevisiae, insect cells in combination withbaculovirus vectors, or cells of a higher organism such as vertebrates,particularly mammals, e.g. COS 7 cells, CHO cells, HEK293 cells, and thelike, may be used as the expression host cells. In some situations, itis desirable to express the gene in eukaryotic cells, where the proteinwill benefit from native folding and post-translational modifications.The polypeptide can then be isolated from cell culture supernatant orfrom cell lysates using affinity chromatography methods or anionexchange/size exclusion chromatography methods, as described above.

With the availability of the protein or fragments thereof in largeamounts, by employing an expression host, the protein may be isolatedand purified in accordance with conventional ways. A lysate may beprepared of the expression host and the lysate purified using HPLC,exclusion chromatography, gel electrophoresis, affinity chromatography,or other purification technique.

Vectors and Host Cells Comprising the Polynucleotides of the Invention

The invention further provides recombinant vectors and host cellscomprising polynucleotides of the invention. In general, recombinantvectors and host cells of the invention are isolated; however, a hostcell comprising a polynucleotide of the invention may be part of agenetically modified animal.

Recombinant vectors. The present invention further provides recombinantvectors (“constructs”) comprising a polynucleotide of the invention.Recombinant vectors include vectors used for propagation of apolynucleotide of the invention, and expression vectors. Vectors usefulfor introduction of the polynucleotide include plasmids and viralvectors, e.g. retroviral-based vectors, adenovirus vectors, etc. thatare maintained transiently or stably in mammalian cells. A wide varietyof vectors can be employed for transfection and/or integration of thegene into the genome of the cells. Alternatively, micro-injection may beemployed, fusion, or the like for introduction of genes into a suitablehost cell.

Expression vectors generally have convenient restriction sites locatednear the promoter sequence to provide for the insertion of nucleic acidsequences encoding heterologous proteins. A selectable marker operativein the expression host may be present. Expression vectors may be usedfor the production of fusion proteins, where the exogenous fusionpeptide provides additional functionality, i.e. increased proteinsynthesis, stability, reactivity with defined antisera, an enzymemarker, e.g. β-galactosidase, etc.

Expression cassettes may be prepared comprising a transcriptioninitiation region, the gene or fragment thereof, and a transcriptionaltermination region. Of particular interest is the use of sequences thatallow for the expression of functional epitopes or domains, usually atleast about 8 amino acids in length, more usually at least about 15amino acids in length, at least about 25 amino acids, at least about 45amino acids, and up to the complete open reading frame of the gene.After introduction of the DNA, the cells containing the construct may beselected by means of a selectable marker, the cells expanded and thenused for expression.

The expression cassettes may be introduced into a variety of vectors,e.g. plasmid, BAC, YAC, bacteriophage such as lambda, P1, M13, etc.,animal or plant viruses, and the like, where the vectors are normallycharacterized by the ability to provide selection of cells comprisingthe expression vectors. The vectors may provide for extrachromosomalmaintenance, particularly as plasmids or viruses, or for integrationinto the host chromosome. Where extrachromosomal maintenance is desired,an origin sequence is provided for the replication of the plasmid, whichmay be low- or high copy-number. A wide variety of markers are availablefor selection, particularly those which protect against toxins, moreparticularly against antibiotics. The particular marker that is chosenis selected in accordance with the nature of the host, where in somecases, complementation may be employed with auxotrophic hosts.Introduction of the DNA construct may use any convenient method, e.g.conjugation, bacterial transformation, calcium-precipitated DNA,electroporation, fusion, transfection, infection with viral vectors,biolistics, etc.

Genetically Modified Cells. The present invention further provides hostcells, which may be isolated host cells, comprising polymorphic BLySnucleic acid molecules of the invention. Suitable host cells includeprokaryotes such as E. coli, B. subtilis, eukaryotes, including insectcells in combination with baculovirus vectors, yeast cells, such asSaccharomyces cerevisiae, or cells of a higher organism such asvertebrates, including amphibians (e.g., Xenopus laevis oocytes), andmammals, particularly humans, e.g. COS cells, CHO cells, HEK293 cells,and the like, may be used as the host cells. Host cells can be used forthe purposes of propagating a polymorphic BLyS nucleic acid molecule,for production of a polymorphic BLyS polypeptide, or in cell-basedmethods for identifying agents which modulate a level of BLyS mRNAand/or protein and/or biological activity in a cell.

Primary or cloned cells and cell lines may be modified by theintroduction of vectors comprising a BLyS gene polymorphism(s). Theisolated polymorphic BLyS nucleic acid molecule may comprise one or morevariant sequences, e.g., a haplotype of commonly occurring combinations.In one embodiment of the invention, a panel of two or more geneticallymodified cell lines, each cell line comprising a BLyS polymorphism, areprovided for substrate and/or expression assays. The panel may furthercomprise cells genetically modified with other genetic sequences,including polymorphisms, particularly other sequences of interest forpharmacogenetic screening, e.g. other genes/gene mutations associatedwith hematologic malignancies, a number of which are known in the art.

Transgenic animals. The subject nucleic acids can be used to generategenetically modified non-human animals or site specific genemodifications in cell lines. The term “transgenic” is intended toencompass genetically modified animals having a deletion or otherknock-out of BLyS gene activity, having an exogenous BLyS gene that isstably transmitted in the host cells, or having an exogenous BLySpromoter operably linked to a reporter gene. Transgenic animals may bemade through homologous recombination, where the BLyS locus is altered.Alternatively, a nucleic acid construct is randomly integrated into thegenome. Vectors for stable integration include plasmids, retrovirusesand other animal viruses, YACs, and the like. Of interest are transgenicmammals, e.g. cows, pigs, goats, horses, etc., and particularly rodents,e.g. rats, mice, etc.

DNA constructs for homologous recombination will comprise at least aportion of a polymorphic BLyS nucleic acid molecule, and will includeregions of homology to the target locus. Conveniently, markers forpositive and negative selection are included. Methods for generatingcells having targeted gene modifications through homologousrecombination are known in the-art. For various techniques fortransfecting mammalian cells, see Known et al. (1990) Methods inEnzymology 185:527-537.

For embryonic stem (ES) cells, an ES cell line may be employed, or EScells may be obtained freshly from a host, e.g. mouse, rat, guinea pig,etc. Such cells are grown on an appropriate fibroblast-feeder layer orgrown in the presence of leukemia inhibiting factor (LIF). When ES cellshave been transformed, they may be used to produce transgenic animals.After transformation, the cells are plated onto a feeder layer in anappropriate medium. Cells containing the construct may be detected byemploying a selective medium. After sufficient time for colonies togrow, they are picked and analyzed for the occurrence of homologousrecombination. Those colonies that show homologous recombination maythen be used for embryo manipulation and blastocyst injection.Blastocysts are obtained from. 4 to 6 week old superovulated females.The ES cells are trypsinized, and the modified cells are injected intothe blastocoel of the blastocyst. After injection, the blastocysts arereturned to each uterine horn of pseudopregnant females. Females arethen allowed to go to term and the resulting litters screened for mutantcells having the construct. By providing for a different phenotype ofthe blastocyst and the ES cells, chimeric progeny can be readilydetected. The chimeric animals are screened for the presence of the BLySgene and males and females having the modification are mated to producehomozygous progeny. The transgenic animals may be any non-human mammal,such as laboratory animals, domestic animals, etc. The transgenicanimals may be used to determine the effect of a candidate drug ontriglyceride synthesis in an in vivo environment.

Preparation of Polymorphic BLyS Polypeptides

In addition to the plurality of uses described in greater detail infollowing sections, the subject nucleic acid compositions find use inthe preparation of all or a portion of the polymorphic BLySpolypeptides, as described above. The subject polynucleotides (includingcDNA or the full-length gene) is used to express a partial or completegene product. Constructs comprising the subject polynucleotides can begenerated synthetically. Alternatively, single-step assembly of a geneand entire plasmid from large numbers of oligodeoxyribonucleotides isdescribed by, e.g., Stemmer et al., Gene (Amsterdam) (1995)164(1):49-53. In this method, assembly PCR (the synthesis of long DNAsequences from large numbers of oligodeoxyribonucleotides (oligos)) isdescribed. The method is derived from DNA shuffling (Stemmer, Nature(1994) 370:389-391), and does not rely on DNA ligase, but instead relieson DNA polymerase to build increasingly longer DNA fragments during theassembly process. Appropriate polynucleotide constructs are purifiedusing standard recombinant DNA techniques as described in, for example,Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., (1989)Cold Spring Harbor Press, Cold Spring Harbor, N.Y., and under currentregulations described in United States Dept. of HHS, National Instituteof Health (NIH) Guidelines for Recombinant DNA Research.

Polynucleotide molecules comprising a polynucleotide sequence providedherein are propagated by placing the molecule in a vector. Viral andnon-viral vectors are used, including plasmids. The choice of plasmidwill depend on the type of cell in which propagation is desired and thepurpose of propagation. Certain vectors are useful for amplifying andmaking large amounts of the desired DNA sequence. Other vectors aresuitable for expression in cells in culture. Still other vectors aresuitable for transfer and expression in cells in a whole animal orperson. The choice of appropriate vector is well within the skill of theart. Many such vectors are available commercially. The partial orfull-length polynucleotide is inserted into a vector typically by meansof DNA ligase attachment to a cleaved restriction enzyme site in thevector. Alternatively, the desired nucleotide sequence can be insertedby homologous recombination in vivo. Typically this is accomplished byattaching regions of homology to the vector on the flanks of the desirednucleotide sequence. Regions of homology are added by ligation ofoligonucleotides, or by polymerase chain reaction using primerscomprising both the region of homology and a portion of the desirednucleotide sequence, for example.

For expression, an expression cassette or system may be employed. Thegene product encoded by a polynucleotide of the invention is expressedin any convenient expression system, including, for example, bacterial,yeast, insect, amphibian and mammalian systems. Suitable vectors andhost cells are described in U.S. Pat. No. 5,654,173. In the expressionvector, an polymorphic BLyS polypeptide-encoding polynucleotide islinked to a regulatory sequence as appropriate to obtain the desiredexpression properties. These can include promoters (attached either atthe 5′ end of the sense strand or at the 3′ end of the antisensestrand), enhancers, terminators, operators, repressors, and inducers.The promoters can be regulated or constitutive. In some situations itmay be desirable to use conditionally active promoters, such astissue-specific or developmental stage-specific promoters. These arelinked to the desired nucleotide sequence using the techniques describedabove for linkage to vectors. Any techniques known in the art can beused. In other words, the expression vector will provide atranscriptional and translational initiation region, which may beinducible or constitutive, where the coding region is operably linkedunder the transcriptional control of the transcriptional initiationregion, and a transcriptional and translational termination region.These control regions may be native to the BLyS gene, or may be derivedfrom exogenous sources.

Expression vectors generally have convenient restriction sites locatednear the promoter sequence to provide for the insertion of nucleic acidsequences encoding heterologous proteins. A selectable marker operativein the expression host may be present. Expression vectors may be usedfor the production of fusion proteins, where the exogenous fusionpeptide provides additional functionality, i.e. increased proteinsynthesis, stability, reactivity with defined antisera, an enzymemarker, e.g. β-galactosidase, etc.

Expression cassettes may be prepared comprising a transcriptioninitiation region, the gene or fragment thereof, and a transcriptionaltermination region. Of particular interest is the use of sequences thatallow for the expression of functional epitopes or domains, usually atleast about 8 amino acids in length, more usually at least about 15amino acids in length, to about 25 amino acids, and up to the completeopen reading frame of the gene. After introduction of the DNA, the cellscontaining the construct may be selected by means of a selectablemarker, the cells expanded and then used for expression.

Polymorphic BLyS polypeptides may be expressed in prokaryotes oreukaryotes in accordance with conventional ways, depending upon thepurpose for expression. For large scale production of the protein, aunicellular organism, such as E. coli, B. subtilis, S. cerevisiae,insect cells in combination with baculovirus vectors, or cells of ahigher organism such as vertebrates, particularly mammals, e.g. COS 7cells, HEK 293, CHO, Xenopus Oocytes, etc., may be used as theexpression host cells. In some situations, it is desirable to express apolymorphic BLyS nucleic acid molecule in eukaryotic cells, where thepolymorphic BLyS protein will benefit from native folding andpost-translational modifications. Small peptides can also be synthesizedin the laboratory. Polypeptides that are subsets of the complete BLySsequence may be used to identify and investigate parts of the proteinimportant for function.

Specific expression systems of interest include bacterial, yeast, insectcell and mammalian cell derived expression systems. Representativesystems from each of these categories is are provided below:

Bacteria. Expression systems in bacteria include those described inChang et al., Nature (1978) 275:615; Goeddel et al., Nature (1979)281:544; Goeddel et al., Nucleic Acids Res. (1980) 8:4057; EP 0 036,776;U.S. Pat. No. 4,551,433; DeBoer et al., Proc. Natl. Acad. Sci. (USA)(1983) 80:21-25; and Siebenlist et al., Cell (1980) 20:269.

Yeast. Expression systems in yeast include those described in Hinnen etal., Proc. Natl. Acad. Sci. (USA) (1978) 75:1929; Ito et al., J.Bacteriol. (1983) 153:163; Kurtz et al., Mol. Cell. Biol. (1986) 6:142;Kunze et al., J. Basic Microbiol. (1985)25:141; Gleeson et al., J. Gen.Microbiol. (1986) 132:3459; Roggenkamp et al., Mol. Gen. Genet. (1986)202:302; Das et al., J. Bacteriol. (1984) 158:1165; De Louvencourt etal., J. Bacteriol. (1983) 154:737; Van den Berg et al., Bio/Technology(1990)8:135; Kunze et al., J. Basic Microbiol (1985)25:141; Cregg etal., Mol. Cell. Biol. (1985) 5:3376; U.S. Pat. Nos. 4,837,148 and4,929,555; Beach and Nurse, Nature (1981) 300:706; Davidow et al., Curr.Genet. (1985) 10:380; Gaillardin et al., Curr. Genet. (1985) 10:49;Ballance et al., Biochem. Biophys. Res. Commun. (1983) 112:284-289;Tilburn et al., Gene (1983) 26:205-221; Yelton et al., Proc. Natl. Acad.Sci. (USA) (1984) 81:1470-1474; Kelly and Hynes, EMBO J. (1985)4:475-479; EP 0 244,234; and WO 91/00357.

Insect Cells. Expression of heterologous genes in insects isaccomplished as described in U.S. Pat. No. 4,745,051; Friesen et al.,“The Regulation of Baculovirus Gene Expression”, in: The MolecularBiology Of Baculoviruses (1986) (W. Doerfler, ed.); EP 0 127,839; EP 0155,476; and Vlak et al., J. Gen. Virol. (1988) 69:765-776; Miller etal., Ann. Rev. Microbiol. (1988) 42:177; Carbonell et al., Gene (1988)73:409; Maeda et al., Nature (1985) 315:592-594; Lebacq-Verheyden etal., Mol. Cell. Biol. (1988) 8:3129; Smith et al., Proc. Natl. Acad.Sci. (USA) (1985) 82:8844; Miyajima et al., Gene (1987) 58:273; andMartin et al., DNA (1988) 7:99. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts aredescribed in Luckow et al., Bio/Technology (1988) 6:47-55, Miller etal., Generic Engineering (1986) 8:277-279, and Maeda et al., Nature(1985) 315:592-594.

Mammalian Cells. Mammalian expression is accomplished as described inDijkema et al., EMBO J. (1985) 4:761, Gorman et al., Proc. Natl. Acad.Sci. (USA) (1982) 79:6777, Boshart et al., Cell (1985) 41:521 and U.S.Pat. No. 4,399,216. Other features of mammalian expression arefacilitated as described in Ham and Wallace, Meth. Enz. (1979) 58:44,Barnes and Sato, Anal. Biochem. (1980) 102:255, U.S. Pat. Nos.4,767,704, 4,657,866, 4,927,762, 4,560,655, WO 90/103430, WO 87/00195,and U.S. Pat. No. RE 30,985.

When any of the above host cells, or other appropriate host cells ororganisms, are used to replicate and/or express the polynucleotides ornucleic acids of the invention, the resulting replicated nucleic acid,RNA, expressed protein or polypeptide, is within the scope of theinvention as a product of the host cell or organism. The product isrecovered by any appropriate means known in the art.

Once the gene corresponding to a selected polynucleotide is identified,its expression can be regulated-in the cell to which the gene is native.For example, an endogenous gene of a cell can be regulated by anexogenous regulatory sequence inserted into the genome of the cell atlocation sufficient to at least enhance expressed of the gene in thecell. The regulatory sequence may be designed to integrate into thegenome via homologous recombination, as disclosed in U.S. Pat. Nos.5,641,670 and 5,733,761, the disclosures of which are hereinincorporated by reference, or may be designed to integrate into thegenome via non-homologous recombination, as described in WO 99/15650,the disclosure of which is herein incorporated by reference. As such,also encompassed in the subject invention is the production of thesubject polymorphic BLyS proteins without manipulation of the encodingnucleic acid itself, but instead through integration of a regulatorysequence into the genome of cell that already includes a gene encodingthe desired protein, as described in the above incorporated patentdocuments.

Antibodies Specific for Polymorphic BLyS Polypeptides

The invention further provides antibodies, particularly isolatedantibodies, that are specific for polymorphic BLyS polypeptides of theinvention. It should be noted that mutations within the protein sequence(unlike rs12583006, rs1224141, and rs12428930 (rs283296)) are generallybelieved required for antibodies specific to the altered proteinsequence to be produced. The antibodies of the invention are useful in avariety of diagnostic assays, as described in further detail below. Forexample, an antibody of the invention can be used to detect apolymorphic BLyS polypeptide in a biological sample.

Isolated polymorphic BLyS polypeptides of the invention are useful forthe production of antibodies, where short fragments provide forantibodies specific for the particular polypeptide, and larger fragmentsor the entire protein allow for the production of antibodies over thesurface of the polypeptide. Accordingly, the invention provides isolatedantibodies which specifically bind a polymorphic BLyS polypeptide, orantigenic fragment thereof. Antibodies may be raised to the wild-type orvariant forms. Antibodies may be raised to isolated peptidescorresponding to these domains, or to the native protein. Antibodies maybe raised to polypeptides and/or peptide fragments of polymorphic BLySfrom any mammalian species.

Particularly useful are antibodies that distinguish between or amongBLyS polymorphic polypeptides. Antibodies may be generated thatspecifically recognize a BLyS polypeptide comprising one or morespecific polymorphisms. Generation of such antibodies, and determinationof their specificity relative to other BLyS polypeptides, is readilyaccomplished by those skilled in the art using conventional methods andassays. As one non-limiting example, an enzyme-linked immunosorbentassay (ELISA) can be used to determine the specificity of a givenmonoclonal antibody for a particular polymorphic BLyS polypeptide.

The polymorphic BLyS polypeptides of the invention are useful for theproduction of antibodies, where short fragments provide for antibodiesspecific for the particular polypeptide, and larger fragments or theentire protein allow for the production of antibodies over the surfaceof the polypeptide. As used herein, the term “antibodies” includesantibodies of any isotype, fragments of antibodies which retain specificbinding to antigen, including, but not limited to, Fab, Fv, scFv, and Fdfragments, fusion proteins comprising such antibody fragments,detectably labeled antibodies, and chimeric antibodies. “Antibodyspecificity”, in the context of antibody-antigen interactions, is a termwell understood in the art, and indicates that a given antibody binds toa given antigen, wherein the binding can be inhibited by that antigen oran epitope thereof which is recognized by the antibody, and does notsubstantially bind to unrelated antigens. Methods of determiningspecific antibody binding are well known to those skilled in the art,and can be used to determine the specificity of antibodies of theinvention for a polymorphic BLyS polypeptide.

Antibodies are prepared in accordance with conventional ways, where theexpressed is polypeptide or protein is used as an immunogen, by itselfor conjugated to known immunogenic carriers, e.g. KLH, pre-S HBsAg,other viral or eukaryotic proteins, or the like. Various adjuvants maybe employed, with a series of injections, as appropriate. For monoclonalantibodies, after one or more booster injections, the spleen isisolated, the lymphocytes immortalized by cell fusion, and then screenedfor high affinity antibody binding. The immortalized cells, i.e.hybridomas, producing the desired antibodies may then be expanded. Forfurther description, see Monoclonal Antibodies: A Laboratory Manual,Harlow and Lane eds., Cold Spring Harbor Laboratories, Cold SpringHarbor, N.Y., 1988. If desired, the mRNA encoding the heavy and lightchains may be isolated and mutagenized by cloning in E. Coli, and theheavy and light chains mixed to further enhance the affinity of theantibody. Alternatives to in vivo immunization as a method of raisingantibodies include binding to phage display libraries, usually inconjunction with in vitro affinity maturation.

Antibodies may be attached, directly or indirectly (e.g., via a linkermolecule) to a solid support for use in a diagnostic assay to determineand/or measure the presence of a polymorphic BLyS polypeptide in abiological sample. Attachment is generally covalent, although it neednot be. Solid supports include, but are not limited to, beads (e.g.,polystyrene beads, magnetic beads, and the like); plastic surfaces(e.g., polystyrene or polycarbonate multi-well plates typically used inan ELISA or radioimmunoassay (RIA), and the like); sheets, e.g., nylon,nitrocellulose, and the like; and chips, e.g., SiO₂ chips such as thoseused in microarrays. Accordingly, the invention further provides assaydevices comprising antibodies attached to a solid support.

A single antibody or a battery of different antibodies can then be usedto create an assay device. Such an assay device can be prepared usingconventional technology known to those skilled in the art. The antibodycan be purified and isolated using known techniques and bound to asupport surface using known procedures. The resulting surface havingantibody bound thereon can be used to assay a test sample, e.g., abiological sample, in vitro to determine if the sample contains one ormore types of BLyS polymorphic polypeptides. For example, antibodieswhich bind only to a specific BLyS polymorphic epitope can be attachedto the surface of a material. Alternatively, a plurality of specificantibodies, which may be arranged in an array, wherein antibodiesspecific for two or more different BLyS polymorphic epitopes areattached to the solid support, can be used. A test sample is broughtinto contact with the antibodies bound to the surface of material.Specific binding can be detected using any known method. If specificbinding is not detected, it can be deduced that the sample does notcontain the specific BLyS polymorphic epitope. As one non-limitingexample of how specific binding can be detected, once the test samplehas been contacted with the antibodies bound to the solid support, asecond, detectably-labeled antibody can be added, which recognizes aBLyS epitope distinct from the epitope recognized by the solidsupport-bound antibody.

A variety of other reagents may be included in the assays to detect BLySpolymorphic polypeptides described herein. These include reagents suchas salts, neutral proteins, e.g. albumin, detergents, etc., that areused to facilitate optimal protein-protein binding, and/or reducenon-specific or background interactions. Reagents that improve theefficiency of the assay, such as protease inhibitors, anti-microbialagents, etc. may be used. The components are added in any order thatprovides for the requisite binding. Incubations are performed at anysuitable temperature, typically between 4° C. and 40° C. Incubationperiods are selected for optimum activity, but may also be optimized tofacilitate rapid high-throughput screening. Typically between 0.1 and 1hours will be sufficient.

Compositions

The present invention further provides compositions, includingpharmaceutical compositions, comprising the polypeptides,polynucleotides, antibodies, recombinant vectors, and host cells of theinvention. These compositions may include a buffer, which is selectedaccording to the desired use of the polypeptide, antibody,polynucleotide, recombinant vector, or host cell, and may also includeother substances appropriate to the intended use. Those skilled in theart can readily select an appropriate buffer, a wide variety of whichare known in the art, suitable for an intended use. In some instances,the composition can comprise a pharmaceutically acceptable excipient, avariety of which are known in the art and need not be discussed indetail herein. Pharmaceutically acceptable excipients have been amplydescribed in a variety of publications, including, for example, A.Gennaro (1995) “Remington: The Science and Practice of Pharmacy”, 19thedition, Lippincott, Williams, & Wilkins.

Diagnostic Assays

Isolated BLyS polymorphic nucleic acid molecules of the invention areuseful in diagnostic assays. The present invention provides diagnosticmethods for detecting, in a nucleic acid sample from an individual, aBLyS polymorphism associated with a condition associated with BLySactivity. The detection methods are useful in methods for identifyingindividuals predisposed to developing a condition associated with BLySactivity, as well as in methods for genetically diagnosing a conditionassociated with BLyS activity.

The invention further provides methods for detecting the presence ofand/or a level of BLyS mRNA in a biological sample; and methods fordetecting the presence of and/or a level of polymorphic BLyS polypeptidein a biological sample.

Thus, in some embodiments, a method is provided for detecting, in apolynucleotide sample derived from an individual, the presence of BLySpolymorphism associated with a disorder associated with BLyS activity inan individual, which method comprises analyzing a polynucleotide samplefrom an individual for the presence of a nucleotide sequencepolymorphism in a BLyS gene, wherein the nucleotide sequencepolymorphism is associated with a condition relating to abnormal fatstorage.

In other embodiments, a method is provide for detecting a level of BLySmRNA in a biological sample derived from an individual, comprisinganalyzing a polynucleotide sample from an individual for the level ofBLyS polypeptide-encoding mRNA. The level of BLyS mRNA may be associatedwith a condition relating to BLyS activity.

In other embodiments, a method is provided for detecting a propensity ofan individual to develop a condition associated with BLyS activity,comprising analyzing a polynucleotide sample derived from the individualfor the presence of a polymorphism in a BLyS gene, wherein saidBLyS-gene polymorphism is associated with a condition associated withBLyS activity.

In other embodiments, a method is provided for genetically diagnosing acondition associated with BLyS activity, comprising analyzing apolynucleotide sample from said individual for the presence of apolymorphism in a BLyS gene, wherein said BLyS gene polymorphism isassociated with a condition associated with BLyS activity.

In still other embodiments, a method is provided for detecting thepresence of and/or the level of a polymorphic BLyS polypeptide in abiological sample, such as a genomic sequence sample. In furtherembodiments, a method is provided for detecting the presence of and/orthe level of a biological activity of a polymorphic BLyS polypeptide ina biological sample.

Polynucleotide samples derived from (e.g., obtained from) an individualare obtained from a biological sample taken from the individual. Anybiological sample that comprises a polynucleotide from the individual issuitable for use in the methods of the invention. The biological samplemay be processed so as to isolate the polynucleotide. Alternatively,whole cells or other biological samples may be used without isolation ofthe polynucleotides contained therein. Detection of a BLyS polymorphismthat is associated with a disorder associated with BLyS activity in apolynucleotide sample derived from an individual can be accomplished byany means known in the art, including, but not limited to, amplificationof a sequence with specific primers; determination of the nucleotidesequence of the polynucleotide sample; hybridization analysis; singlestrand conformational polymorphism analysis; denaturing gradient gelelectrophoresis; mismatch cleavage detection; and the like. Detection ofa BLyS polymorphism that is associated with a condition associated withBLyS activity can also be accomplished by detecting an alteration in thelevel of a mRNA transcript of a BLyS gene; aberrant modification of aBLyS gene, e.g., an aberrant methylation pattern; the presence of anon-wild-type splicing pattern of BLyS mRNA; an alteration in the levelof BLyS polypeptide; and/or an alteration in BLyS polypeptide biologicalactivity.

Detection of a BLyS polymorphism by analyzing a polynucleotide samplecan be conducted in a number of ways. A test nucleic acid sample can-beamplified with primers which amplify a sequence region known to comprisea BLyS polymorphism(s). Non-limiting examples of such sequences areprovided in Example 1. Genomic DNA or mRNA can be used directly.Alternatively, the region of interest can be cloned into a suitablevector and grown in sufficient quantity for analysis. The nucleic acidmay be amplified by conventional techniques, such as a polymerase chainreaction (PCR), to provide sufficient amounts for analysis. The use ofthe polymerase chain reaction is described in a variety of publications,including, e.g., “PCR Protocols (Methods in Molecular Biology)” (2000)J. M. S. Bartlett and D. Stirling, eds, Humana Press; and “PCRApplications: Protocols for Functional Genomics” (1999) Innis, Gelfand,and Sninsky, eds., Academic Press. Once the region comprising a BLySpolymorphism has been amplified, the BLyS polymorphism can be detectedin the PCR product by nucleotide sequencing, by SSCP analysis, or anyother method known in the art. In performing SSCP analysis, the PCRproduct may be digested with a restriction endonuclease that recognizesa sequence within the PCR product generated by using as a template areference BLyS sequence, but does not recognize a corresponding PCRproduct generated by using as a template a variant BLyS sequence byvirtue of the fact that the variant sequence no longer contains arecognition site for the restriction endonuclease.

PCR may also be used to determine whether a polymorphism is present byusing a primer that is specific for the polymorphism. Such methods maycomprise the steps of collecting from an individual a biological samplecomprising the individual's genetic material as template, optionallyisolating template nucleic acid (genomic DNA, mRNA, or both) from thebiological sample, contacting the template nucleic acid sample with oneor more primers that specifically hybridize with a BLyS polymorphicnucleic acid molecule under conditions such that hybridization andamplification of the template nucleic acid molecules in the sampleoccurs, and detecting the presence, absence, and/or relative amount ofan amplification product and comparing tie length to a control sample.Observation of an amplification product of the expected size is anindication that the BLyS polymorphism contained within the BLySpolymorphic primer is present in the test nucleic acid sample.Parameters such as hybridization conditions, BLyS polymorphic primerlength, and position of the polymorphism within the BLyS polymorphicprimer may be chosen such that hybridization will not occur unless apolymorphism present in the primer(s) is also present in the samplenucleic acid. Those of ordinary skill in the art are well aware of howto select and vary such parameters. See, e.g., Saiki et al. (1986)Nature 324:163; and Saiki et al (1989) Proc. Natl. Acad. Sci. USA86:6230. As one non-limiting example, a PCR primer comprising the T78Cvariation described in Example 1 may be used.

Alternatively, various methods are known in the art that utilizeoligonucleotide ligation as a means of detecting polymorphisms. See,e.g., Riley et al. (1990) Nucleic Acids Res. 18:2887-2890; and Delahuntyet al. (1996) Am. J Hum. Genet. 58:1239-1246.

A detectable label may be included in an amplification reaction.Suitable labels include fluorochromes, e.g. fluorescein isothiocyanate(FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin,6-carboxyfluorescein (6-FAM),2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE),6-carboxy-X-rhodamine (ROX),6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein(5-FAM) or N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), radioactivelabels, e.g. ³²P, ³⁵S, ³H; etc. The label may be a two stage system,where the amplified DNA is conjugated to biotin, haptens, etc. having ahigh affinity binding partner, e.g. avidin, specific antibodies, etc.,where the binding partner is conjugated to a detectable label. The labelmay be conjugated to one or both of the primers. Alternatively, the poolof nucleotides used in the amplification is labeled, so as toincorporate the label into the amplification product.

The sample nucleic acid may be sequenced by a dideoxy chain terminationmethod or other well-known methods. Genomic DNA or mRNA may be useddirectly. If mRNA is used, a cDNA copy may first be made. If desired,the sample nucleic acid can be amplified using a PCR. A variety ofsequencing reactions known in the art can be used to directly sequencethe BLyS gene, or a portion thereof in which a specific polymorphism isknown to occur, and detect polymorphisms by comparing the sequence ofthe sample nucleic acid with a reference polynucleotide that contains aBLyS polymorphism. Any of a variety of automated sequencing procedurescan be used. See, e.g., WO 94/16101; Cohen et al. (1996) Adv.Chromatography 36:127-162.

Hybridization with the variant sequence may also be used to determinethe presence of a BLyS polymorphism. Hybridization analysis can becarried out in a number of different ways, including, but not limited toSouthern blots, Northern blots, dot blots, microarrays, etc. Thehybridization pattern of a control and variant sequence to an array ofoligonucleotide probes immobilized on a solid support, as described inU.S. Pat. No. 5,445,934, or in WO 95/35505, may also be used as a meansof detecting the presence of variant sequences. Identification of apolymorphism in a nucleic acid sample can be performed by hybridizing asample and control nucleic acids to high density arrays containinghundreds or thousands of oligonucleotide probes. Cronin et al. (1996)Human Mutation 7:244-255; and Kozal et al. (1996) Nature Med. 2:753-759.

Single strand conformational polymorphism (SSCP) analysis; denaturinggradient gel electrophoresis (DGGE); mismatch cleavage detection; andheteroduplex analysis in gel matrices can also be used to detectpolymorphisms. Alternatively, where a polymorphism creates or destroys arecognition site for a restriction endonuclease (restriction fragmentlength polymorphism, RFLP), the sample is digested with thatendonuclease, and the products size fractionated to determine whetherthe fragment was digested. Fractionation is performed by gel orcapillary electrophoresis, particularly acrylamide or agarose gels. Theaforementioned techniques are well known in the art. Detaileddescription of these techniques can be found in a variety ofpublications, including, e.g., “Laboratory Methods for the Detection ofMutations and Polymorphisms in DNA” (1997) G. R. Taylor, ed., CRC Press,and references cited therein.

A number of methods are available for determining the expression levelof a polymorphic BLyS nucleic acid molecule, e.g., a polymorphic BLySmRNA, or polymorphic BLyS polypeptide in a particular sample. Diagnosismay be performed by a number of methods to determine the absence orpresence or altered amounts of normal or abnormal BLyS mRNA in a patientsample. For example, detection may utilize staining of cells orhistological sections with labeled antibodies, performed in accordancewith conventional methods. Cells are permeabilized to stain cytoplasmicmolecules. The antibodies of interest are added to the cell sample, andincubated for a period of time sufficient to allow binding to theepitope, usually at least about 10 minutes. The antibody may be labeledith radioisotopes, enzymes, fluorescers, chemiluminescers, or otherlabels for direct detection. Alternatively, a second stage antibody orreagent is used to amplify the signal. Such reagents are well known inthe art. For example, the primary antibody may be conjugated to biotin,with horseradish peroxidase-conjugated avidin added as a second stagereagent. Alternatively, the secondary antibody conjugated to afluorescent compound, e.g. fluorescein, rhodamine, Texas red, etc. Finaldetection uses a substrate that undergoes a color change in the presenceof the peroxidase. The absence or presence of antibody binding may bedetermined by various methods, including flow cytometry of dissociatedcells, microscopy, radiography, scintillation counting, etc. Thepresence and/or the level of a polymorphic BLyS polypeptide may also bedetected and/or quantitated in any way known to one of ordinary skill.

In addition, a test can include measurements of the expression of BLySmRNA. Biochemical studies may be performed to determine whether asequence polymorphism in a BLyS coding region or control regions isassociated with disease. Disease associated polymorphisms may includedeletion or truncation of the gene, mutations that alter expressionlevel, that affect the activity of the protein, etc.

Changes in the promoter or enhancer sequence that may affect expressionlevels of BLyS can be compared to expression levels of the normal alleleby various methods known in the art. Methods for determining promoter orenhancer strength include quantitation of the expressed natural protein;insertion of the variant control element into a vector with a reportergene such as β-galactosidase, luciferase, chloramphenicolacetyltransferase, etc. that provides for convenient quantitation; andthe like.

Screening for mutations in a polymorphic BLyS polypeptide may be basedon the functional or antigenic characteristics of the protein. Proteintruncation assays are useful in detecting deletions that may affect thebiological activity of the protein. Various immunoassays designed todetect polymorphisms in polymorphic BLyS polypeptides may be used inscreening. Where many diverse genetic mutations lead to a particulardisease phenotype, functional protein assays have proven to be effectivescreening tools. The activity of the encoded a polymorphic BLySpolypeptide may be determined by comparison with a reference BLySpolypeptide lacking a specific polymorphism.

Diagnostic methods of the subject invention in which the level of BLySgene expression is of interest will typically involve comparison of theBLyS nucleic acid abundance of a sample of interest with that of acontrol value to determine any relative differences, where thedifference may be measured qualitatively and/or quantitatively, whichdifferences are then related to the presence or absence of an abnormalBLyS gene expression pattern. A variety of different methods fordetermine the nucleic acid abundance in a sample are known to those ofskill in the art, where particular methods of interest include thosedescribed in: Pietu et al., Genome Res. (June 1996) 6: 492-503; Zhao etal., Gene (Apr. 24, 1995) 156: 207-213; Soares, Curr. Opin. Biotechnol.(October 1997) 8: 542-546; Raval, J. Pharmacol Toxicol Methods (November1994) 32: 125-127; Chalifour et al., Anal. Biochem (Feb. 1, 1994) 216:299-304; Stolz & Tuan, Mol. Biotechnol. (December 19960 6: 225-230; Honget al., Bioscience Reports (1982) 2: 907; and McGraw, Anal. Biochem.(1984) 143: 298. Also of interest are the methods disclosed in WO97/27317, the disclosure of which is herein incorporated by reference.

Additional tests that have been associated with NHL disease severity orprogression can be combined with the polymorphism test described aboveto render a full diagnosis or outlook result. One test that can becombined with the polymorphism test is a measurement of tumor cell orserum BLyS levels (Novak et al. Blood 2002, supra). Among other teststhat can be combined are, positron emission tomography with2-[18F]fluororo-2-deoxy-D-glucose (FDG-PET) (see Mikhaeel et al., Ann.Oncol. 2005 16:1514-23); fine needle cytology (on its own or combinedwith immunocytochemistry, flow cytometry, Southern blot analysis,polymerase chain reaction and fluorescent in situ hybridization (FISH))(see Dey, Cytopathology, 2006 17:275-87); and Ga-67 scintigraphy (seeIsrael et al., Cancer, 2002 94:873-878.)

Monitoring Effects of Drug Treatment

Monitoring the influence of agents (e.g., drugs, compounds) on theexpression or activity of a BLyS protein (e.g., modulation oftranscriptional activation) can be applied not only in basic drugscreening, but also in clinical trials. For example, the effectivenessof an agent determined by a screening assay as described herein toincrease BLyS gene expression, protein levels, or upregulate BLySactivity, can be monitored in clinical trials of subjects exhibitingdecreased BLyS gene expression, protein levels, or down-regulated BLySactivity. Alternatively, the effectiveness of an agent determined by ascreening assay to decrease BLyS gene expression, protein levels, ordownregulate BLyS activity, can be monitored in clinical trials ofsubjects exhibiting increased BLyS gene expression, protein levels, orupregulated BLyS activity. In such clinical trials, the expression oractivity of a BLyS gene, and preferably, other genes that have beenimplicated in, for example, a disorder associated with BLyS activity canbe used as a “read out” or markers of the phenotype of a particularcell.

For example, and not by way of limitation, genes, including BLyS, thatare modulated in cells by treatment with an agent (e.g., compound, drugor small molecule) which modulates BLyS activity (e.g., identified in ascreening assay as described herein) can be identified. Thus, to studythe effect of agents on fat storage disorders, for example, in aclinical trial, cells can be isolated and RNA prepared and analyzed forthe levels of expression of BLyS and other genes implicated in adisorder associated with BLyS activity. The levels of gene expression(i.e., a gene expression pattern) can be quantified by Northern blotanalysis or RT-PCR, as described herein, or alternatively by measuringthe amount of protein produced, by one of the methods as describedherein, or by measuring the levels of activity of BLyS or other genes.In this way, the gene expression pattern can serve as a marker,indicative of the physiological response of the cells to the agent.Accordingly, this response state may be determined before, and atvarious points during treatment of the individual with the agent.

In some embodiments, the present invention provides a method formonitoring the effectiveness of treatment of a subject with an agent(e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleicacid, small molecule, or other drug that modifies a BLyS activity)comprising the steps of (i) obtaining a pre-administration sample from asubject prior to administration of the agent; (ii) detecting the levelof expression of a BLyS protein, mRNA, or genomic DNA in thepre-administration sample; (iii) obtaining one or morepost-administration samples from the subject, (iv) detecting the levelof expression or activity of the BLyS protein, mRNA, or genomic DNA inthe post-administration samples; (v) comparing the level of expressionor activity of the BLyS protein, mRNA, or genomic DNA in thepre-administration sample with the BLyS protein, mRNA, or genomic DNA inthe post administration sample or samples; and (vi) altering theadministration of the agent to the subject accordingly. For example,increased administration of the agent may be desirable to increase theexpression or activity of BLyS to higher levels than detected, i.e., toincrease the effectiveness of the agent. Alternatively, decreasedadministration of the agent may be desirable to decrease expression oractivity of BLyS to lower levels than detected, i.e. to decrease theeffectiveness of the agent. According to such an embodiment, BLySexpression or activity may be used as an indicator of the effectivenessof an agent, even in the absence of an observable phenotypic response.

Linkage Analysis: Diagnostic screening may be performed forpolymorphisms that are genetically linked to a phenotypic variant inBLyS activity or expression, particularly through the use ofmicrosatellite markers or single nucleotide polymorphisms (SNP). Themicrosatellite or SNP polymorphism itself may not phenotypicallyexpressed, but is linked to sequences that result in altered activity orexpression. Two polymorphic variants may be in linkage disequilibrium,i.e. where alleles show non-random associations between genes eventhough individual loci are in Hardy-Weinberg equilibrium.

Linkage analysis may be performed alone, or in combination with directdetection of phenotypically evident polymorphisms. The use ofmicrosatellite markers for genotyping is well documented. For examples,see Mansfield et al. (1994) Genomics 24:225-233; and Ziegle et al.(1992) Genomics 14:1026-10331. The use of SNPs for genotyping isillustrated in Underhill et al. (1996) Proc. Natl. Acad. Sci. USA93:196-200.

Genetic linkage maps show the relative locations of specific DNA markersalong a chromosome. Any inherited physical or molecular characteristicthat differs among individuals and is easily detectable in thelaboratory is a potential genetic marker. DNA sequence polymorphisms areuseful markers because they are plentiful and easy to characterizeprecisely. Many such polymorphisms are located in non-coding regions anddo not affect the phenotype of the organism, yet they are detectable atthe DNA level and can be used as markers. Examples include restrictionfragment length polymorphisms (RFLPs), which reflect sequence variationsin DNA sites or differences in the length of the product, which can becleaved by DNA restriction enzymes, microsatellite markers, which areshort repeated sequences that vary in the number of repeated units,single nucleotide polymorphisms (SNPs), and the like.

The “linkage” aspect of the map is a measure of how frequently twomarkers are inherited together. The closer the markers are to each otherphysically, the less likely a recombination event will fall between andseparate them. Recombination frequency thus provides an estimate of thedistance between two markers. The value of the genetic map is that aninherited trait can be located on the map by following the inheritanceof a DNA marker present in affected individuals, but absent inunaffected individuals, even though the molecular basis for the traitmay not yet be understood. Genetic maps have been used to find the exactchromosomal location of several important disease genes, includingcystic fibrosis, muscular dystrophy, sickle cell disease, Tay-Sachsdisease, fragile X syndrome and many others.

An emerging class of marker for genetic analysis of the singlenucleotide polymorphism, and other simple polymorphisms, e.g. deletions,double nucleotide polymorphisms, etc. SNPs are generally biallelicsystems, that is, there are two alleles that a population may have forany particular marker. This means that the information content per SNPmarker is relatively low when compared to microsatellite markers, whichmay have upwards of 10 alleles. SNPs also tend to be verypopulation-specific; a marker that is polymorphic in one population maynot be very polymorphic in another.

SNP markers offer a number of benefits that will make them anincreasingly valuable tool. SNPs, found approximately every kilobase(see Wang et al. (1998) Science 280:1077-1082), offer the potential forgenerating very high density genetic maps, which will be extremelyuseful for developing haplotyping systems for genes or regions ofinterest, and because of the nature of SNPs, they may in fact be thepolymorphisms associated with the disease phenotypes under study. Thelow mutation rate of SNPs also makes them excellent markers for studyingcomplex genetic traits.

Substrate screening assay. Substrate screening assays are used todetermine the catalytic activity of a BLyS protein or peptide fragmenton a substrate. Many suitable assays are known in the art, including theuse of primary or cultured cells, genetically modified cells (e.g.,where DNA encoding the BLyS polymorphism to be studied is introducedinto the cell within an artificial construct), cell-free systems, e.g.microsomal preparations or recombinantly produced enzymes in a suitablebuffer, or in animals, including human clinical trials.

Typically a detectably labeled substrate is input into the assay system,and the generation of labeled triglyceride is measured over time. Thechoice of detection system is determined by the substrate and thespecific assay parameters. Assays are conventionally run, and willinclude negative and positive controls, varying concentrations ofsubstrate and enzyme, etc. Exemplary assays may be found in theliterature, as described above.

Pharmacokinetic parameters. Pharmacokinetic parameters providefundamental data for designing safe and effective dosage regimens. Adrug's volume of distribution, clearance, and the derived parameter,half-life, are particularly important, as they determine the degree offluctuation between a maximum and minimum plasma concentration during adosage interval, the magnitude of steady state concentration and thetime to reach steady state plasma concentration upon chronic dosing.Parameters derived from in vivo drug administration are useful indetermining the clinical effect of a particular BLyS genotype.

Expression assay. An assay to determine the effect of a sequencepolymorphism on BLyS expression. Expression assays may be performed incell-free extracts, or by transforming cells with a suitable vector.Alterations in expression may occur in the basal level that is expressedin one or more cell types, or in the effect that an expression modifierhas on the ability of the gene to be inhibited or induced. Expressionlevels of a variant alleles are compared by various methods known in theart. Methods for determining promoter or enhancer strength includequantitation of the expressed natural protein; insertion of the variantcontrol element into a vector with a reporter gene such asgalactosidase, luciferase, chloramphenicol acetyltransferase, etc. thatprovides for convenient quantitation; and the like.

Gel shift or electrophoretic mobility shift assay provides a simple andrapid method for detecting DNA-binding proteins (Ausubel, F. M. et al.(1989) In: Current Protocols in Molecular Biology, Vol. 2, John Wileyand Sons, New York). This method has been used widely in the study ofsequence-specific DNA-binding proteins, such as transcription factors.The assay is based on the observation that complexes of protein and DNAmigrate through a nondenaturing polyacrylamide gel more slowly than freeDNA fragments or double-stranded oligonucleotides. kilo The gel shiftassay is performed by incubating a purified protein, or a complexmixture of proteins (such as nuclear or cell extract preparations), withan end-labeled DNA fragment containing the putative protein bindingsite. The reaction products are then analyzed on a nondenaturingpolyacrylamide gel. The specificity of the DNA-binding protein for theputative binding site is established by competition experiments usingDNA fragments or oligonucleotides containing a binding site for theprotein of interest, or other unrelated DNA sequences.

Expression assays can be used to detect differences in expression ofpolymorphisms with respect to tissue specificity, expression level, orexpression in response to exposure to various substrates, and/or timingof expression during development.

Genotyping BLyS genotyping is performed by DNA or RNA sequence and/orhybridization analysis of any convenient sample from a patient, e.g.biopsy material, blood sample (serum, plasma, etc.), buccal cell sample,etc. A nucleic acid sample from an individual is analyzed for thepresence of polymorphisms in BLyS, particularly those that affect theactivity or expression of BLyS. Specific sequences of interest includeany polymorphism that leads to changes in basal expression in one ormore tissues, to changes in the modulation of BLyS expression bymodifiers, or alterations in BLyS substrate specificity and/or activity.

The effect of a polymorphism in the BLyS gene sequence on the responseto a particular substrate or modifier of BLyS is determined by in vitroor in vivo assays. Such assays may include monitoring the metabolism ofa substrate during clinical trials to determine the BLyS biologicalactivity, specificity or expression level. Generally, in vitro assaysare useful in determining the direct effect of a particularpolymorphism, while clinical studies will also detect an biologicalphenotype that is genetically linked to a polymorphism.

The response of an individual to the substrate or modifier can then bepredicted by determining the BLyS genotype, with respect to thepolymorphism. Where there is a differential distribution of apolymorphism by racial background, guidelines for drug administrationcan be generally tailored to a particular ethnic group.

The basal expression level in different tissue may be determined byanalysis of tissue samples from individuals typed for the presence orabsence of a specific polymorphism. Any convenient method may be use,e.g. ELISA, RIA, etc. for protein quantitation, northern blot or otherhybridization analysis, quantitative RT-PCR, etc. for mRNA quantitation.The tissue specific expression is correlated with the genotype.

The alteration of BLyS expression in response to a modifier isdetermined by administering or combining the candidate modifier with anexpression system, e.g. animal, cell, in vitro transcription assay, etc.The effect of the modifier on BLyS transcription and/or steady statemRNA levels is determined. As with the basal expression levels, tissuespecific interactions are of interest. Correlations are made between theability of an expression modifier to affect BLyS activity, and thepresence of the provided polymorphisms. A panel of different modifiers,cell types, etc. may be screened in order to determine the effect undera number of different conditions.

A BLyS polymorphism that results in altered biological activity orspecificity is determined by a variety of assays known in the art. Theligand may be tested for formation of triglyceride product in vitro, forexample in defined buffer, or in cell or subcellular lysates, where theability of a substrate to be acted on by BLyS under physiologicconditions is determined. Where there are not significant issues oftoxicity from the substrate or products(s), in vivo human trials maybeutilized, as previously described.

The genotype of an individual is determined with respect to the providedBLyS gene polymorphisms. The genotype is useful for determining thepresence of a phenotypically evident polymorphism, and for determiningthe linkage of a polymorphism to phenotypic change.

Any of a number of techniques known to those skilled in the art can beused to detect a polymorphism in a BLyS gene, using an isolatedpolynucleotide of the invention. These include, but are not limited to,direct sequencing of the interval from affected individuals (Chadwick etal. (1996) Biotechniques 20:676-683); and hybridization with one or moreprobes derived from a region of a BLyS gene, including allele-specificoligonucleotide hybridization (Wong and Senadheera (1997) Clin. Chem.43:1857-1861). The region being detected can optionally be amplified byknown techniques, including, but not limited to, a polymerase chainreaction. Other analytical techniques include, but are not limited to,single-strand conformation analysis; restriction length polymorphism(RFLP) analysis; enzymatic mismatch cleavage techniques such asglycosylase mediated polymorphism detection (Vaughan and McCarthy (1998)Nucl. Acids Res. 26:810-815); heteroduplex PCR (Deuter and Muller (1998)Hum. Mutat. 11:84-89); and fiberoptic DNA sensor array techniques(Healey et al. (1997) Anal. Biochem. 251:270-279). Automated methods ofdetecting polymorphisms have been developed and can be used in themethods of the present invention. See, for example, Marshall and Hodgson(1998) Nature Biotechnol 16:27-31. Other methods include, for example,PCR-RFLP. Hani et al. (1998) J. Clin. Invest. 101:521-526.

Treatment Methods

The present invention hither provides a method of treating an individualclinically diagnosed with a condition associated with BLyS activity. Themethods generally comprises analyzing a polynucleotide sample from anindividual clinically diagnosed with a condition associated with BLySactivity for the presence or absence of a BLyS gene polymorphism. Thepresence of a BLyS gene polymorphism associated with a conditionrelating to abnormal hematological cell growth confirms the clinicaldiagnosis of a condition associated with BLyS activity. A treatment planthat is most effective for individuals clinically diagnosed as having acondition associated with BLyS activity is then selected on the basis ofthe detected BLyS polymorphism. Genotype information obtained asdescribed above can be used to predict the response of the individual toa particular BLyS substrate (e.g., activator or inhibitor of BLySbiological activity), or modifier of BLyS gene expression. Thus, theinvention further provides a method for predicting a patient'slikelihood to respond to a drug treatment for a condition associatedwith BLyS activity, comprising determining a patient's genotype in aBLyS gene, wherein the presence of a BLyS allele associated with acondition associated with BLyS activity is predictive of the patient'slikelihood to respond to a drug treatment for the condition. Where anexpression modifier inhibits BLyS expression, then drugs that are a BLySsubstrate will be metabolized more slowly if the modifier isco-administered. Where an expression modifier induces BLyS expression, aco-administered substrate will typically be metabolized more rapidly.Similarly, changes in BLyS activity will affect the metabolism of anadministered drug. The pharmacokinetic effect of the interaction willdepend on the metabolite that is produced, e.g. a prodrug is metabolizedto an active form, a drug is metabolized to an inactive form, anenvironmental compound is metabolized to a toxin, etc. Consideration isgiven to the route of administration, drug-drug interactions, drugdosage, etc.

Thus, another aspect of the invention provides methods for tailoring anindividual's prophylactic, or therapeutic treatment with BLyS expressionand/or activity modulators according to that individual's drug responsegenotype. Pharmacogenomics allows a clinician or physician to targetprophylactic or therapeutic treatments to patients who will most benefitfrom the treatment and to avoid treatment of patients who willexperience toxic drug-related side effects.

Agents that have a stimulatory or inhibitory effect on BLyS expressionlevels or BLyS biological activity can be administered to individuals totreat (prophylactically or therapeutically) disorders associated withBLyS activity. Additionally, the isolated polymorphic BLyS nucleic acidmolecules of the present invention, as well as agents, or modulatorswhich have a stimulatory or inhibitory effect on BLyS expression levelsor BLyS biological activity can be administered to individuals to treata condition associated with BLyS activity. Differences in metabolism oftherapeutics can lead to severe toxicity or therapeutic failure byaltering the relation between dose and blood concentration of thepharmacologically active drug. Thus, a physician or clinician mayconsider applying knowledge obtained in relevant pharmacogenomicsstudies in determining whether to administer a modulator of BLySexpression or biological activity (“a BLyS modulator”) as well astailoring the dosage and/or therapeutic regimen of treatment with a BLySmodulator.

Determination of how a given BLyS polymorphism is predictive of apatient's likelihood of responding to a given drug treatment for acondition relating to abnormal fat storage can be accomplished bydetermining the genotype of the patient in the BLyS gene, as describedabove, and/or determining the effect of the drug on BLyS geneexpression, and/or determining the effect of the drug on BLyS biologicalactivity. Information generated from one or more of these approaches canbe used to determine appropriate dosage and treatment regimens forprophylactic or therapeutic treatment an individual. This knowledge,when applied to dosing or drug selection, can avoid adverse reactions ortherapeutic failure and thus enhance therapeutic or prophylacticefficiency when treating a subject with a BLyS molecule or BLySmodulator, such as a modulator identified by one of the exemplaryscreening assays described herein.

Microarrays

The invention further provides an array of oligonucleotides (alsoreferred to herein as “probes”), where discrete positions on the arrayare complementary to one or more of the provided polymorphic sequences,e.g. oligonucleotides of at least 12 nt, at least about 15 nt, at leastabout 18 nt, at least about 20 nt, or at least about 25 nt, or longer,and including the sequence flanking the polymorphic position. Such anarray may comprise a series of oligonucleotides, each of which canspecifically hybridize to a different polymorphism. For examples ofarrays, see Hacia et al. (1996) Nat. Genet. 14:441-447 and DeRisi et al.(1996) Nat. Genet. 14:457-460.

An array may include all or a subset of the polymorphisms listed above.One or more polymorphic forms may be present in the array. In someembodiments, an array includes at least 2 different polymorphicsequences, i.e. polymorphisms located at unique positions within thelocus, and may include as many all of the provided polymorphisms. Arraysof interest may further comprise sequences, including polymorphisms, ofother genetic sequences, particularly other sequences of interest forpharmacogenetic screening, including, but not limited to, other genesassociated with hematological malignancies, including but not limited tothose associated with B-CLL such as zap-70, CD38 etc., or an equivalentthereof in another species. The oligonucleotide sequence on the array isgenerally at least about 12 nt in length, at least about 15 nt, at leastabout 18 nt, at least about 20 nt, or at least about 25 nt, or may bethe length of the provided polymorphic sequences, or may extend into theflanking regions to generate fragments of 100 to 200 nt in length. Forexamples of arrays, see Ramsay (1998) Nature Biotech. 16:40-44; Hacia etal (1996) Nature Genetics 14:441-447; Lockhart et al (1996) NatureBiotechnol. 14:1675-1680; and De Risi et al. (1996) Nature Genetics14:457-460.

A number of methods are available for creating microarrays of biologicalsamples, such as arrays of DNA samples to be used in DNA hybridizationassays. Exemplary are PCT Application Serial. No. WO95/35505, publishedDec. 28, 1995; U.S. Pat. No. 5,445,934, issued Aug. 29, 1995; andDrnanac et al. (1993) Science 260:1649-1652. Yershov et al, (1996)Genetics 93:4913-4918 describe an alternative construction of anoligonucleotide array. The construction and use of oligonucleotidearrays is reviewed by Ramsay (1998) supra.

Methods of using high density oligonucleotide arrays are known in theart. For example, Milosavljevic et al. (1996) Genomics 37:77-86 describeDNA sequence recognition by hybridization to short oligomers. See also,Drmanac et al. (1998) Nature Biotech. 16:54-58; and Drnanac and Drmanac(1999) Methods Enzymol 303:165-178; The use of arrays for identificationof unknown mutations is proposed by Ginot (1997) Human Mutation 10:1-10.

Detection of known mutations is described in Hacia et al. (1996) Nat.Genet. 14:441-447; Cronin et al. (1996) Human Mut. 7:244-255; andothers. The use of arrays in genetic mapping is discussed in Chee et al.(1996) Science 274:610-613; Sapolsky and Lishutz (1996) Genomics33:445-456; etc. Shoemaker et al. (1996) Nat. Genet. 14:450-456 performquantitative phenotypic analysis of yeast deletion mutants using aparallel bar-coding strategy.

Quantitative monitoring of gene expression patterns with a complementaryDNA microarray is described in Schena et al. (1995) Science 270:467.DeRisi et al (1997) Science 270:680-686 explore gene expression on agenomic scale. Wodicka et al. (1997) Nat. Biotech. 15:1-15 performgenome wide expression monitoring in S. cerevisiae.

A DNA sample is prepared in accordance with conventional methods, e.g.lysing cells, removing cellular debris, separating the DNA fromproteins, lipids or other components present in the mixture and thenusing the isolated DNA for cleavage. See Molecular Cloning, A LaboratoryManual, 2nd ed. (eds. Sambrook et al.) CSH Laboratory Press, Cold SpringHarbor, N.Y. 1989. Generally, at least about 0.5 μg of DNA will beemployed, usually at least about 5 μg of DNA, while less than 50 μg ofDNA will usually be sufficient.

The nucleic acid samples are cleaved to generate probes. It will beunderstood by one of skill in the art that any method of random cleavagewill generate a distribution of fragments, varying in the average sizeand standard deviation. Usually the average size will be at least about12 nucleotides in length, more usually at least about 20 nucleotides inlength, and preferably at least about 35 nucleotides in length. Wherethe variation in, size is great, conventional methods may be used toremove the large and/or small regions of the fragment population.

It is desirable, but not essential to introduce breaks randomly, with amethod which does not act preferentially on specific sequences.Preferred methods produce a reproducible pattern of breaks. Methods forintroducing random breaks or nicks in nucleic acids include reactionwith Fenton reagent to produce hydroxyl radicals and other chemicalcleavage systems, integration mediated by retroviral integrase, partialdigestion with an ultra-frequent cutting restriction enzymes, partialdigestion of single stranded with S1 nuclease, partial digestion withDNAse I in the absence or presence of Mn⁺⁺, etc.

The fragmented nucleic acid samples are denatured and labeled. Labelingcan be performed according to methods well known in the art, using anymethod that provides for a detectable signal either directly orindirectly from the nucleic acid fragment. In a preferred embodiment,the fragments are end-labeled, in order to minimize the steric effectsof the label. For example, terminal transferase may be used to conjugatea labeled nucleotide to the nucleic acid fragments. Suitable labelsinclude biotin and other binding moieties; fluorochromes, e.g.fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin,allophycocyanin, 6-carboxyfluorescein (6-FAM),2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE),6-carboxy-X-rhodamine (ROX),6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein(5-FAM) or N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), and thelike. Where the label is a binding moiety, the detectable label isconjugated to a second stage reagent, e.g. avidin, streptavidin, etc.that specifically binds to the binding moiety, for example a fluorescentprobe attached to streptavidin. Incorporation of a fluorescent labelusing enzymes such as reverse transcriptase or DNA polymerase, prior tofragmentation of the sample, is also possible.

Each of the labeled genome samples is separately hybridized to an arrayof oligonucleotide probes. Hybridization of the labeled sequences isaccomplished according to methods well known in the art. Hybridizationcan be carried out under conditions varying in stringency, preferablyunder conditions of high stringency, e.g. 6×SSPE, at 65° C., to allowfor hybridization of complementary sequences having extensive homology,usually having no more than one or two mismatches in a probe of 25nucleotides in length, i.e. at least 95% to 100% sequence identity.

High density microarrays of oligonucleotides are known in the art andare commercially available. The sequence of oligonucleotides on thearray will correspond to the known target sequences of one of thegenomes, as previously described. Arrays of interest for the subjectmethods will generally comprise at least about 10³ different sequences,usually at least about 10⁴ different sequences, and may comprise 10⁵ ormore different sequences. The length of oligonucleotide present on thearray is an important factor in how sensitive hybridization will be tothe presence of a mismatch. Usually oligonucleotides will be at leastabout 12 nt in length, more usually at least about 15 nt in length,preferably at least about 20 nt in length and more preferably at leastabout 25 nt in length, and will be not longer than about 35 nt inlength, usually not more than about 30 nt in length.

Methods of producing large arrays of oligonucleotides are described inU.S. Pat. No. 5,134,854 (Pirrung et al.), and U.S. Pat. No. 5,445,934(Fodor et al.) using light-directed synthesis techniques. Using acomputer controlled system, a heterogeneous array of monomers isconverted, through simultaneous coupling at a number of reaction sites,into a heterogeneous array of polymers. Alternatively, microarrays aregenerated by deposition of pre-synthesized oligonucleotides onto a solidsubstrate, for example as described in International Patent applicationWO 95/35505.

Microarrays can be scanned to detect hybridization of the labeled genomesamples. Methods and devices for detecting fluorescently marked targetson devices are known in the art. Generally such detection devicesinclude a microscope and light source for directing light at asubstrate. A photon counter detects fluorescence from the substrate,while an x-y translation stage varies the location of the substrate. Aconfocal detection device that may be used in the subject methods isdescribed in U.S. Pat. No. 5,631,734. A scanning laser microscope isdescribed in Shalon et al. (1996) Genome Res. 6:639. A scan, using theappropriate excitation line, is performed for each fluorophore used. Thedigital images generated from the scan are then combined for subsequentanalysis. For any particular array element, the ratio of the fluorescentsignal from one Nucleic acid sample is compared to the fluorescentsignal from the other Nucleic acid sample, and the relative signalintensity determined.

Methods for analyzing the data collected by fluorescence detection areknown in the art. Data analysis includes the steps of determiningfluorescent intensity as a function of substrate position from the datacollected, removing outliers, i.e. data deviating from a predeterminedstatistical distribution, and calculating the relative binding affinityof the targets from the remaining data. The resulting data may bedisplayed as an image with the intensity in each region varyingaccording to the binding affinity between targets and probes.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric.

Example 1 Search for SNPs Associated with Increased Risk of DevelopingNHL

We genotyped 9 tagSNPs within the BLyS gene in a clinic-based study of441 incident Caucasian NHL cases and 475 frequency matched Caucasiancontrols seen at the Mayo Clinic from 2002-2005. We evaluated theassociation of individual SNPs as well as haplotypes from the BLyS genewith risk of NHL. We also jointly tested the main effects for allindependent (r²<0.25) SNPs using a multivariate logistic regression(MLR) model. As a secondary analysis, for those SNPs showing significantresults (<5% significance), we evaluated associations between those whohad all high risk alleles at the SNPs of interest compared to those whohad all low risk alleles at the SNPs of interest. Additionally, weevaluated serum BLyS levels by ELISA in these same subjects. Serum BLySlevels were determined by ELISA.

Results: In the individual single SNP logistic regression analysis, 3 ofthe 9 tagSNPs were significant at p<0.05. Those three SNPs have beenidentified as the major alleles of following: rs12583006 (SEQ ID NO:5),rs1224141 (SEQ ID NO:6), and rs12428930 (rs283296) (SEQ ID NO:7).Haplotype and MLR results were nonsignificant. However, when wecategorized participants into low and high risk groups based on riskalleles at the three statistically significant SNPs, we found the highrisk variant (i.e., the presence of all three major alleles) had an oddsratio (OR) of 2.086 (p=0.0001) for risk of B-cell NHL. When the analysiswas restricted by histologic subtype we found that diffuse large B-celllymphoma or follilcular lymphoma grade 3 had an OR of 3.163 (p=0.01),follicular lymphoma grade I/II had an OR=2.547 (p=0.046), and chroniclymphocytic leukemia (CLL) had an OR=1.238 (p=0.13). Because there wasnot a significant correlation of the high risk variant with CLL, weperformed an additional analysis in which we included all B cell NHLcases excluding CLL and the OR was 2.799 (p=0.0001).

Example 2 Measurement of BLyS Levels Inpatients with Patients with Highand Low Risk Variant Alleles

We next wanted to determine if serum BLyS levels correlated with eitherthe high or low risk variant. The mean serum BLyS level in thoseindividuals (untreated cases and controls) who carried the low riskvariant at all three SNPs was significantly lower (p=0.0061) at 1.3ng/ml (n=25, range: undetectable-4.4 ng/ml) compared to 4.3 ng/ml inthose with the high risk variant (n=74, range: undetectable-66.8 ng/ml).

BLyS levels were determined using a BLyS ELISA as follows. ELISA plateswere coated with 1 μg/ml anti-BLyS (ZymoGenetics, Seattle, Wash.) andBLyS was detected with 1 μg/ml biotinylated anti-BLyS (ZymoGenetics,Seattle, Wash.) followed by streptavidin-HRP and TMB substrate asdescribed in Novak et al. Blood 2004, supra. Patient serum samples werediluted 1:5 and 1:25 in triplicate and BLyS serum levels were calculatedfrom a standard curve generated with recombinant human BLyS(ZymoGenetics, Seattle, Wash.) in 20% human sera. The detection limit ofpurified BLyS was 300 pg/ml.

Conclusions: In summary, we have found that genetic variation in theBLyS gene is significantly associated with an increased risk ofdeveloping B-cell NHL, particularly follicular and large B-celllymphoma. Additionally, this increased lymphoma risk is associated withan increase in serum BLyS levels.

While the present invention has been described with reference to thespecific embodiments thereof, it is to be understood by those skilled inthe art that various changes may be made and an equivalence may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the object, spirit and scope of the present invention.All such modifications are intended to be within the scope of the claimsappended hereto.

1. A method of detecting a propensity of a human to develop a conditionselected from the group consisting of B-cell non-Hodgkin's lymphoma,diffuse large B-cell lymphoma, follicular lymphoma grade 1, follicularlymphoma grade 2, and follicular lymphoma grade 3, wherein said methodcomprises: (a) detecting in a polynucleotide sample derived from saidhuman the presence of a polymorphism in a B-lymphocyte stimulator (BLyS)gene, wherein said polymorphism is selected from the group consisting ofa T allele at rs12583006, a T allele at rs122414, and an A allele atrs1248930, and (b) classifying said human as having an increasedpropensity to develop said condition if said polymorphism is present. 2.The method of claim 1, wherein said method comprises detecting in saidpolynucleotide sample the presence of two or more polymorphisms selectedfrom said group, and classifying said human as having an increasedpropensity to develop said condition if said two or more polymorphismsare present.
 3. The method of claim 2, wherein said method comprisesdetecting in said polynucleotide sample the presence of threepolymorphisms selected from said group and classifying said human ashaving an increased propensity to develop said condition if said threepolymorphisms are present.
 4. The method of claim 1, wherein thepresence of said polymorphism is determined by contacting apolynucleotide from said human with a polynucleotide probe which iscapable of hybridizing to said polymorphism under stringent conditions;and determining whether hybridization has occurred, thereby indicatingthe presence of said polymorphism.
 5. A method for identifying a humanhaving an increased risk of developing a condition selected from thegroup consisting of B-cell non-Hodgkin's lymphoma, diffuse large B-celllymphoma, follicular lymphoma grade 1, follicular lymphoma grade 2, andfollicular lymphoma grade 3, wherein said method comprises: (a)detecting the presence of a polymorphism selected from the groupconsisting of a T allele at rs12583006, a T allele at rs122414, and an Aallele at rs1248930 in a polynucleotide sample from said human, and (b)classifying said human as having increased risk of developing saidcondition based on the presence of said polymorphism.
 6. The method ofclaim 4, wherein said method comprises detecting the presence of two ormore polymorphisms selected from said group in said polynucleotidesample, and classifying said human as having increased risk ofdeveloping said condition when said two or more polymorphisms arepresent.
 7. The method of claim 6, wherein said method comprisesdetecting the presence of three polymorphisms selected from said groupin said polynucleotide sample, and classifying said human as havingincreased risk of developing said condition when said threepolymorphisms are present.
 8. The method of claim 4, wherein thepresence of said polymorphism is detected by contacting saidpolynucleotide sample with a polynucleotide probe which is capable ofhybridizing to said polymorphism under stringent conditions; anddetermining that hybridization has occurred, thereby indicating thepresence of said polymorphism.